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    QIAGEN Proteinase K
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    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
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    19131
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    QIAGEN Proteinase K
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    Qiagen pbs buffer
    QIAGEN Proteinase K
    For protease digestion during DNA and RNA preparation Kit contents Qiagen Proteinase K 2mL 600mAU mL Activity Ready to use Solution For Protease Digestion During DNA and RNA Preparation Offer Broad Substrate Specificity with High Activity for a Wide Range of Reaction Conditions Used in Most DNA and RNA Isolation
    https://www.bioz.com/result/pbs buffer/product/Qiagen
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    Images

    1) Product Images from "Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa"

    Article Title: Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02861-5

    O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P

    Techniques Used: Ex Vivo, Purification, Produced

    In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P

    Techniques Used: In Situ, Staining

    2) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.
    Figure Legend Snippet: Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.

    Techniques Used: Inhibition, Activation Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.
    Figure Legend Snippet: Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.

    Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    3) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    4) Product Images from "HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury"

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2017.12.004

    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.
    Figure Legend Snippet: Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Techniques Used: Activity Assay, Western Blot, Staining

    5) Product Images from "Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays"

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays

    Journal: BMC Infectious Diseases

    doi: 10.1186/s12879-015-0903-2

    IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.
    Figure Legend Snippet: IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.

    Techniques Used: Binding Assay, Western Blot, Stripping Membranes, Acrylamide Gel Assay, Incubation

    6) Product Images from "Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity"

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02753

    The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p
    Figure Legend Snippet: The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Techniques Used: Cell Culture, Incubation, Infection, Staining

    7) Product Images from "Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *"

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.590976

    Secondary screen. A, schematic representation of the RT-PCR in vitro splicing assay. 20-μl splicing reaction was incubated in the presence of 500 μ m compound at 30 °C for 90 min before the splicing reaction was stop by heat inactivation. After proteinase K digestion, the spliced and unspliced RNA is amplified by RT-PCR. B shows examples of compounds that either inhibit pre-mRNA splicing or do not interfere with the splicing reaction. The star indicates splicing reactions that were inhibited. Lane M , marker (hyperladder, 1 kb).
    Figure Legend Snippet: Secondary screen. A, schematic representation of the RT-PCR in vitro splicing assay. 20-μl splicing reaction was incubated in the presence of 500 μ m compound at 30 °C for 90 min before the splicing reaction was stop by heat inactivation. After proteinase K digestion, the spliced and unspliced RNA is amplified by RT-PCR. B shows examples of compounds that either inhibit pre-mRNA splicing or do not interfere with the splicing reaction. The star indicates splicing reactions that were inhibited. Lane M , marker (hyperladder, 1 kb).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Splicing Assay, Incubation, Amplification, Marker

    8) Product Images from "RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues"

    Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167336

    Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).
    Figure Legend Snippet: Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis

    9) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Comparison of DNA extraction protocols in spiked sputum samples. The  M. tuberculosis mc 2 7000  stock suspension was diluted and used to spike negative sputum samples. Box plots with C T  median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ®  method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    10) Product Images from "Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro"

    Article Title: Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamem.2014.07.026

    Effect of proteins on exosome association. PC3-derived exosomes were treated with proteinase K to remove surface protein moieties. PC3-derived exosomes (untreated or subjected to proteinase K) were incubated with PC3 and MCF-7 cells for 4 hrs at 37°C.
    Figure Legend Snippet: Effect of proteins on exosome association. PC3-derived exosomes were treated with proteinase K to remove surface protein moieties. PC3-derived exosomes (untreated or subjected to proteinase K) were incubated with PC3 and MCF-7 cells for 4 hrs at 37°C.

    Techniques Used: Derivative Assay, Incubation

    11) Product Images from "An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis"

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33541-5

    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Figure Legend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Techniques Used: Incubation

    12) Product Images from "An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis"

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33541-5

    Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.
    Figure Legend Snippet: Fe 0 -corroding activities in filtrates of an OS7 culture. Filtrates of a culture of strain OS7 grown for 10 days under H 2 + CO 2 (80:20) were prepared, and 1 ml of the filtrate was added to 20 ml of basal medium containing Fe 0 granules either without treatment (OS7 filtrate), or with proteinase K treatment at 50 °C for 20 min and at 100 °C for 5 min (OS7 filtrate + K), or with treatment at 50 °C for 20 min (OS7 filtrate + 50 °C). As a control, 21-ml basal medium containing Fe 0 granules was treated with proteinase K at 50 °C for 20 min and at 100 °C for 5 min (Control + K). Duplicate samples of each treatment were incubated at 37 °C for 5, 8, 11 and 14 days, and the amounts of Fe 2+ in basal medium (A) , and amounts of H 2 in headspace gas (B) were determined. The amounts of CH 4 in headspace gas were also determined, and were zero in all samples.

    Techniques Used: Incubation

    13) Product Images from "Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs "

    Article Title: Functional Overlap but Lack of Complete Cross-Complementation of Streptococcus mutans and Escherichia coli YidC Orthologs

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01366-07

    Membrane insertion of F 1 F o ATPase subunits a and c. (A) Membrane insertion of subunit c of the F 1 F o ATPase. Proteinase K digestion of c-10h-tag at the periplasmic side generates a slightly smaller protein. JS7131 expressing 247YidC1, 247YidC2, or YidC1/YidC2
    Figure Legend Snippet: Membrane insertion of F 1 F o ATPase subunits a and c. (A) Membrane insertion of subunit c of the F 1 F o ATPase. Proteinase K digestion of c-10h-tag at the periplasmic side generates a slightly smaller protein. JS7131 expressing 247YidC1, 247YidC2, or YidC1/YidC2

    Techniques Used: Expressing

    14) Product Images from "Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase"

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01271-19

    STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).
    Figure Legend Snippet: STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).

    Techniques Used: Overlay Assay

    15) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    16) Product Images from "Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa"

    Article Title: Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00081-15

    OprF is a potential C3 binding acceptor on the P. aeruginosa surface. (A) Mid-log-phase P. aeruginosa was incubated with proteinase K (PK) at 37°C, washed with PBS, and incubated with 20% normal human serum. C3 binding to the bacterial surface
    Figure Legend Snippet: OprF is a potential C3 binding acceptor on the P. aeruginosa surface. (A) Mid-log-phase P. aeruginosa was incubated with proteinase K (PK) at 37°C, washed with PBS, and incubated with 20% normal human serum. C3 binding to the bacterial surface

    Techniques Used: Binding Assay, Incubation

    17) Product Images from "Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability"

    Article Title: Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.6b00489

    Membrane topology and post-translational modifications of PRCD. A , Osmotically intact discs were treated with membrane impermeable proteinase K (at 8 μg/ml) followed by Western blotting with antibodies against PRCD and rhodopsin (recognizing its
    Figure Legend Snippet: Membrane topology and post-translational modifications of PRCD. A , Osmotically intact discs were treated with membrane impermeable proteinase K (at 8 μg/ml) followed by Western blotting with antibodies against PRCD and rhodopsin (recognizing its

    Techniques Used: Western Blot

    18) Product Images from "The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ"

    Article Title: The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00929

    Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p
    Figure Legend Snippet: Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Techniques Used: Polymerase Chain Reaction, Incubation, Isolation

    19) Product Images from "Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase"

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01271-19

    STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).
    Figure Legend Snippet: STYM1 cellular extracts have inhibitory protein. Extract was spotted onto plates, allowed to dry under aerobic conditions, and overlaid with a soft top agar inoculated with P. gingivalis . Plates were imaged 2 days after anaerobic growth. (A) Agar overlay assay with equal protein amounts of soluble cell extracts from STYM1, SYB13, SYB7, and 11842. (B to D) Agar overlay assays with STYM1 cellular extract either treated with proteinase K (Prot K) or heat-killed proteinase K (Heat-killed Prot K) (B), passage through a 10-kDa-MWCO filter (Flow and Conc.) (C), or heat treatment (95°C for 20 min) (D).

    Techniques Used: Overlay Assay

    20) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    21) Product Images from "Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B"

    Article Title: Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B

    Journal: mBio

    doi: 10.1128/mBio.00206-15

    Silicibacter sp. strain TrichCH4B biological response to NO and Trichodesmium erythraeum . (A) Exogenous NO increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. With increasing amounts of NO, Silicibacter sp. TrichCH4B formed more biofilm, as quantified by crystal violet staining (OD 570 ). Biofilm formation was normalized against growth (OD 600 ). (B) Addition of T. erythraeum spent medium (TSM) increases Silicibacter sp. TrichCH4B NO formation. Silicibacter sp. TrichCH4B was grown anaerobically with TSM as described in Materials and Methods. To fully digest any proteins, TSM was also treated with proteinase K before the addition to Silicibacter sp. TrichCH4B. Only the TSM (high-molecular-weight [MW] fraction, or retentate, from a 5-kDa-MWCO membrane filter) showed stimulation of NO formation by Silicibacter sp. TrichCH4B. (C) TSM addition increases SiliNOS gene expression. Silicibacter sp. TrichCH4B was grown aerobically with various amounts of TSM, and cDNA from Silicibacter sp. TrichCH4B mRNA was prepared as described in Materials and Methods. Expression of the SiliNOS gene (ΔC(t)) was calculated using Bio-Rad CFX Manager software with rpoD as a reference gene. SiliNOS gene expression increased with the amount of TSM added. (D) TSM addition increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. Silicibacter sp. TrichCH4B biofilm formation increased with the amount of TSM added, as quantified by crystal violet staining. Values that are significantly different are indicated by bars and asterisks as follows: *, P
    Figure Legend Snippet: Silicibacter sp. strain TrichCH4B biological response to NO and Trichodesmium erythraeum . (A) Exogenous NO increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. With increasing amounts of NO, Silicibacter sp. TrichCH4B formed more biofilm, as quantified by crystal violet staining (OD 570 ). Biofilm formation was normalized against growth (OD 600 ). (B) Addition of T. erythraeum spent medium (TSM) increases Silicibacter sp. TrichCH4B NO formation. Silicibacter sp. TrichCH4B was grown anaerobically with TSM as described in Materials and Methods. To fully digest any proteins, TSM was also treated with proteinase K before the addition to Silicibacter sp. TrichCH4B. Only the TSM (high-molecular-weight [MW] fraction, or retentate, from a 5-kDa-MWCO membrane filter) showed stimulation of NO formation by Silicibacter sp. TrichCH4B. (C) TSM addition increases SiliNOS gene expression. Silicibacter sp. TrichCH4B was grown aerobically with various amounts of TSM, and cDNA from Silicibacter sp. TrichCH4B mRNA was prepared as described in Materials and Methods. Expression of the SiliNOS gene (ΔC(t)) was calculated using Bio-Rad CFX Manager software with rpoD as a reference gene. SiliNOS gene expression increased with the amount of TSM added. (D) TSM addition increases Silicibacter sp. TrichCH4B biofilm formation. Static biofilm assays were performed as described in Materials and Methods. Silicibacter sp. TrichCH4B biofilm formation increased with the amount of TSM added, as quantified by crystal violet staining. Values that are significantly different are indicated by bars and asterisks as follows: *, P

    Techniques Used: Staining, Molecular Weight, Expressing, Software

    22) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    23) Product Images from "Plasmid-normalized quantification of relative mitochondrial DNA copy number"

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33684-5

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Figure Legend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Techniques Used: Incubation, Lysis

    24) Product Images from "Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth ▿"

    Article Title: Identification of Amino Acid Residues in BK Virus VP1 That Are Critical for Viability and Growth ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01316-07

    Release and infectivity of mutants. Vero cells were transfected with 5 μg of linear WT or mutant DNA. (A) Five days later, medium supernatant was treated with DNase I, proteinase K, and VP1 DNA amplified via PCR. This revealed that these mutants
    Figure Legend Snippet: Release and infectivity of mutants. Vero cells were transfected with 5 μg of linear WT or mutant DNA. (A) Five days later, medium supernatant was treated with DNase I, proteinase K, and VP1 DNA amplified via PCR. This revealed that these mutants

    Techniques Used: Infection, Transfection, Mutagenesis, Amplification, Polymerase Chain Reaction

    25) Product Images from "Capsular Polysaccharide Is a Receptor of a Clostridium perfringens Bacteriophage CPS1"

    Article Title: Capsular Polysaccharide Is a Receptor of a Clostridium perfringens Bacteriophage CPS1

    Journal: Viruses

    doi: 10.3390/v11111002

    CPS1 binds to polysaccharides on the cell surface. ( A ) CPS1 adsorption assay with ATCC 13124. CPS1 was infected at 30 °C. It was found that 95% of CPS1 adsorbed in 40 min. ( B ) CPS1 adsorption assay with various treatment. 1: BHI (No cells), 2: untreated cells, 3: acetate treatment, 4: 10 mM periodate treatment, 5: 100 mM periodate treatment, 6: proteinase K treatment.
    Figure Legend Snippet: CPS1 binds to polysaccharides on the cell surface. ( A ) CPS1 adsorption assay with ATCC 13124. CPS1 was infected at 30 °C. It was found that 95% of CPS1 adsorbed in 40 min. ( B ) CPS1 adsorption assay with various treatment. 1: BHI (No cells), 2: untreated cells, 3: acetate treatment, 4: 10 mM periodate treatment, 5: 100 mM periodate treatment, 6: proteinase K treatment.

    Techniques Used: Adsorption, Infection

    26) Product Images from "Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products"

    Article Title: Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

    Journal: Human Gene Therapy Methods

    doi: 10.1089/hgtb.2015.013

    Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the  SV40  sequence was used to spike crude ( left ) and HPLC-purified ( middle ) AAV8 samples and AAV2 ( right ) samples before HPLC purification at a vector concentration of 6.25×10 13  copies/ml; qPCR analysis was performed, targeting spike-specific  SV40  (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown ( n =3). Asterisks indicate statistical significance between crude and purified samples, where * p
    Figure Legend Snippet: Effects of enzymatic treatments on qPCR analysis. A lentiviral vector plasmid carrying the SV40 sequence was used to spike crude ( left ) and HPLC-purified ( middle ) AAV8 samples and AAV2 ( right ) samples before HPLC purification at a vector concentration of 6.25×10 13 copies/ml; qPCR analysis was performed, targeting spike-specific SV40 (hatched columns) and vector-specific sequences in AAV8 (gray columns) and AAV2 (cross-hatched columns); DNA copy number was determined on the basis of calibration curves generated from circular plasmid DNA standards. NT, no treatment; DT, treatment with Benzonase and DNase I; DT/PK, treatment with Benzonase and DNase I followed by proteinase K. Mean data are shown ( n =3). Asterisks indicate statistical significance between crude and purified samples, where * p

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Sequencing, High Performance Liquid Chromatography, Purification, Concentration Assay, Generated

    27) Product Images from "DNA Polymerase Beta Participates in Mitochondrial DNA Repair"

    Article Title: DNA Polymerase Beta Participates in Mitochondrial DNA Repair

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00237-17

    DNA polymerase β is detected in the mitochondria. (A) Mouse mitochondria purified from liver and brain were digested with increasing amounts of proteinase K to remove outer membrane-bound proteins. The liver samples (left) show no Polβ in the mitochondrial compartment. The brain (right) mitochondrial sample revealed Polβ after proteinase digestion, evidence that Polβ was inside the mitochondrial membrane. VDAC1 was used as a mitochondrial outer membrane marker and was digested at 0.5 mg/ml proteinase. Lamin A/C was used as a marker for nuclear contamination. GAPDH shows the extent of cytoplasmic contamination. A second antibody was also used to verify the results [Polβ (Rb)]. Fifty micrograms of protein was loaded. (B) The kidney preparations had a clear Polβ band present. Human kidney cells (HEK293T) were used to determine whether Polβ was found in the mitochondria of human cells and whether the levels could be artificially manipulated. Vector control HEK293T preparations had visible amounts of endogenous Polβ present. Overexpression of Polβ caused elevated levels of mitochondrial Polβ. The TFAM mitochondrial control antibody detected only the human form of the protein. (C) To verify Polβ antibody specificity, HEK293T cells were modified for Polβ knockout using CRISPR/cas9. The far left lane shows the molecular mass of purified Polβ protein. Total cellular protein extracts from clones 23 and 24 have no remaining Polβ. The red line indicates a molecular mass marker at 39 kDa. (D) Mitochondrial extracts from HEK293T/Polβ-KO cells compared to parental and parental+Cas9 cells. Extracts were exposed to either 0.05 or 0.1 mg/ml proteinase K or had treatment (NT). Polβ was detected in the extracts from the parental cell lines but were absent in Polβ-KO lines (clones 23 and 24). Red lines indicate molecular mass markers at 39 and 52 kDa. (E) To visualize Polβ in the mitochondria, HEK293T cells were transfected with either a C-terminal GFP or FLAG full-length (FL) Polβ, a positive control with an MLS sequence from the mitochondrial protein SOD2, and the FL Polβ protein with a 17-aa N-terminal deletion (D17N) (see Materials and Methods for a full description). (F) Immunoblot analysis of modified Polβ-GFP constructs to verify protein localization. Samples 1 to 4, mitochondrial extracts. Samples 5 to 8, whole-cell lysates. Lanes 1 and 5, mock-transfected cells. Lanes 2 and 6, FL Polβ-GFP. Lanes 3 and 7, SOD2 MLS–Polβ-GFP. Lanes 4 and 8, D17N-Polβ-GFP. VDAC1 was used to show mitochondrial enrichment of the samples. Tubulin was used as a cytoplasmic marker. Lanes 1 to 4 contained 10 μg mitochondrial extract per lane, and lanes 5 to 8 contained 2 × 10 4 cells per lane. (G) Localization of the C-terminal FLAG constructs depicted in panel E by immunofluorescence. The FL Polβ-FLAG construct is detected in the mitochondria (PDM channel). Addition of the SOD2 MLS signal enhanced the PDM signal; however, only with the deletion of the 17-aa N-terminal region (D17N) was there complete loss of nuclear localization and enhanced mitochondrial localization (also see Fig. S1G in the supplemental material). DAPI was used to visualize the nucleus (magnification, ×63, z-stack; scale bar = 20 μm).
    Figure Legend Snippet: DNA polymerase β is detected in the mitochondria. (A) Mouse mitochondria purified from liver and brain were digested with increasing amounts of proteinase K to remove outer membrane-bound proteins. The liver samples (left) show no Polβ in the mitochondrial compartment. The brain (right) mitochondrial sample revealed Polβ after proteinase digestion, evidence that Polβ was inside the mitochondrial membrane. VDAC1 was used as a mitochondrial outer membrane marker and was digested at 0.5 mg/ml proteinase. Lamin A/C was used as a marker for nuclear contamination. GAPDH shows the extent of cytoplasmic contamination. A second antibody was also used to verify the results [Polβ (Rb)]. Fifty micrograms of protein was loaded. (B) The kidney preparations had a clear Polβ band present. Human kidney cells (HEK293T) were used to determine whether Polβ was found in the mitochondria of human cells and whether the levels could be artificially manipulated. Vector control HEK293T preparations had visible amounts of endogenous Polβ present. Overexpression of Polβ caused elevated levels of mitochondrial Polβ. The TFAM mitochondrial control antibody detected only the human form of the protein. (C) To verify Polβ antibody specificity, HEK293T cells were modified for Polβ knockout using CRISPR/cas9. The far left lane shows the molecular mass of purified Polβ protein. Total cellular protein extracts from clones 23 and 24 have no remaining Polβ. The red line indicates a molecular mass marker at 39 kDa. (D) Mitochondrial extracts from HEK293T/Polβ-KO cells compared to parental and parental+Cas9 cells. Extracts were exposed to either 0.05 or 0.1 mg/ml proteinase K or had treatment (NT). Polβ was detected in the extracts from the parental cell lines but were absent in Polβ-KO lines (clones 23 and 24). Red lines indicate molecular mass markers at 39 and 52 kDa. (E) To visualize Polβ in the mitochondria, HEK293T cells were transfected with either a C-terminal GFP or FLAG full-length (FL) Polβ, a positive control with an MLS sequence from the mitochondrial protein SOD2, and the FL Polβ protein with a 17-aa N-terminal deletion (D17N) (see Materials and Methods for a full description). (F) Immunoblot analysis of modified Polβ-GFP constructs to verify protein localization. Samples 1 to 4, mitochondrial extracts. Samples 5 to 8, whole-cell lysates. Lanes 1 and 5, mock-transfected cells. Lanes 2 and 6, FL Polβ-GFP. Lanes 3 and 7, SOD2 MLS–Polβ-GFP. Lanes 4 and 8, D17N-Polβ-GFP. VDAC1 was used to show mitochondrial enrichment of the samples. Tubulin was used as a cytoplasmic marker. Lanes 1 to 4 contained 10 μg mitochondrial extract per lane, and lanes 5 to 8 contained 2 × 10 4 cells per lane. (G) Localization of the C-terminal FLAG constructs depicted in panel E by immunofluorescence. The FL Polβ-FLAG construct is detected in the mitochondria (PDM channel). Addition of the SOD2 MLS signal enhanced the PDM signal; however, only with the deletion of the 17-aa N-terminal region (D17N) was there complete loss of nuclear localization and enhanced mitochondrial localization (also see Fig. S1G in the supplemental material). DAPI was used to visualize the nucleus (magnification, ×63, z-stack; scale bar = 20 μm).

    Techniques Used: Purification, Marker, Plasmid Preparation, Over Expression, Modification, Knock-Out, CRISPR, Clone Assay, Transfection, Positive Control, Sequencing, Construct, Immunofluorescence

    28) Product Images from "Plasmid-normalized quantification of relative mitochondrial DNA copy number"

    Article Title: Plasmid-normalized quantification of relative mitochondrial DNA copy number

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33684-5

    Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.
    Figure Legend Snippet: Time curve showing mtDNA content measured in a blood sample extracted by BioRobot EZ1 Advanced after incubation using 4 different lysis buffers. The buffers used were G2 (included in the EZ1 Tissue kit) and Pierce RIPA Buffer Lyse (Thermo Fisher Scientific, Waltham, MA, USA). Both buffers were used pure and in combination with Proteinase K (PK). The baseline is obtained using the standard EZ1 tissue kit protocol without incubation.

    Techniques Used: Incubation, Lysis

    29) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Overexpression of STAT3 restrains susceptibility to KSHV lytic activation. (A to D) BCBL-1 cells were transfected with the pEGFPN1 plasmid (GFP) or the pEGFPN1-STAT3 plasmid (STAT3-GFP), exposed to VPA after 12 h, and harvested after another 24 h for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and K8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method (A), for determination of relative amounts of cell-associated KSHV DNA by qPCR (B), for determination of K8.1 + cells by flow cytometry (C), or assayed for infectious virions in the cell supernatant by inoculation of HUVECs and staining with anti-LANA antibody and DAPI 48 h later (D). (A and B) Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) Numbers indicate the percentages of GFP + (i.e., transfected) cells that were lytic. Lytic (i.e., K8.1 + ) gates were placed based on a 1% cutoff in similarly treated cells that were stained with isotype control antibody. A representative of the results of two experiments is shown. (E) BCBL-1 cells were transfected with GFP plasmid or STAT3-GFP plasmid (as in panels A to D) and harvested at 24 h posttransfection for Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Over Expression, Activation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.
    Figure Legend Snippet: Chemical inhibition of STAT3 results in KSHV lytic activation, increase in the number of lytic cells, and increase in production of infectious virions. BCBL-1 cells were treated with VPA, WP1066, or VPA plus WP1066 or left untreated. Cells were harvested at 1, 6, 12, and 18 h posttreatment (A), at 24 h posttreatment (B), and at 48 h posttreatment (C). (A) RNA was analyzed at different times by qRT-PCR for relative transcript levels of 4 lytic genes, ORF50 , ORF59 , ORF9 , and ORFK8.1 , compared to untreated cells. KSHV-specific transcript levels were normalized to GAPDH , HRPTI , and B2M (β2 microglobulin), and fold changes were determined by the ΔΔ C T method. Data are presented as means and SEM and are representative of the results of two separate experiments with 3 technical replicates. (B) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. Numbers indicate the percent K8.1 + (lytic) cells; these percentages were determined after comparison with similarly treated cells stained with isotype control antibody. (C) Cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification.

    Techniques Used: Inhibition, Activation Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Activation Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.
    Figure Legend Snippet: Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot

    Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.
    Figure Legend Snippet: Cells expressing high levels of STAT3 protein are refractory to spontaneous and induced KSHV lytic activation. BCBL-1 cells were treated with TPA or VPA or left untreated (U). Cells were harvested at 1, 24, and 48 h (A and B) and at 48 h (C) posttreatment. (A) RNA was isolated and subjected to qRT-PCR to determine the relative levels of lytic ORFK8.1 transcripts. Fold changes were calculated by the ΔΔ C T method, normalized to three housekeeping genes, GAPDH , HPRTI , and B2M (β2 microglobulin). The data are presented as means and standard errors of the mean (SEM) and are representative of the results of two experiments with 3 technical replicates. (B) Cell lysates were immunoblotted using antibodies to K8.1 and β-actin. (C) Cells were immunostained for K8.1 and STAT3 and subjected to flow cytometry. The numbers within the dot plots in the left column indicate the percent K8.1 hi cells under untreated or TPA- or VPA-treated conditions after comparison with similarly treated cells stained with isotype control antibody; the dashed oval gates indicate K8.1 lo cells. The numbers within the dot plots in the three middle columns indicate the percent K8.1 hi cells in subpopulations expressing different levels of STAT3, i.e., STAT3 hi (high), STAT3 int (intermediate), and STAT3 lo (low). Numbers in the dot plots on the right indicate the percent K8.1 lo cells (from the oval gates in the left-hand dot plots) expressing low, intermediate, and high levels of STAT3.

    Techniques Used: Expressing, Activation Assay, Isolation, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    30) Product Images from "STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus"

    Article Title: STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

    Journal: Journal of Virology

    doi: 10.1128/JVI.02008-15

    Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.
    Figure Legend Snippet: Knockdown of endogenous STAT3 results in increased spontaneous KSHV lytic activation. (A) BCBL-1 cells were transfected with scrambled siRNA or two different siRNAs to STAT3 [si STAT3 (1) and si STAT3 (2)] and harvested 18 h and 48 h later for determination of relative amounts of transcripts from KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (B) BCBL-1 cells were transfected with scrambled siRNA (Sc) or siRNAs to STAT3 (si-1 and si-2) and harvested 48 h later for determination of relative amounts of cell-associated KSHV DNA by qPCR. Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. (C) BCBL-1 cells were transfected with either STAT3 siRNA and FITC + scrambled siRNA (to mark transfected cells) at a 3:1 ratio [si STAT3 (1)] or with FITC − and FITC + scrambled siRNA at a 3:1 ratio (Scrambled). After 36 h, the FITC + (i.e., transfected) population was examined by flow cytometry for K8.1 + cells. Numbers indicate percentages of transfected cells that were spontaneously lytic. A representative of the results of two experiments is shown. (D) BCBL-1 cells were treated as for panel B, and cell supernatants were used to inoculate primary HUVECs. After 48 h, HUVECs were fixed, permeabilized, stained with anti-LANA antibody and DAPI, and visualized at ×40 magnification. (E) BCBL-1 cells were transfected with scrambled siRNA or siRNA to STAT3 , harvested at 48 h posttransfection, and subjected to Western blot analysis using anti-STAT3 and anti-β-actin antibodies.

    Techniques Used: Activation Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Staining, Western Blot

    Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.
    Figure Legend Snippet: Knockdown of STAT3 suppresses KAP1, and suppression of KAP1 causes an increase in the levels of lytic transcripts. BCBL-1 cells were transfected with scrambled siRNA (Sc) or two different siRNAs to STAT3 [ STAT3 (1) and STAT3 (2)] (A and B) or scrambled siRNA (Sc) or siRNA to KAP1 (C and D) and harvested 24 h (C and D) or 48 h (A and B) later for determination of the relative amounts of transcripts from cellular KAP1 (A) and the KSHV lytic genes ORF50 , ORF59 , ORF9 , and ORFK8.1 (C) by qRT-PCR after normalization to 18S rRNA using the ΔΔ C T method or subjected to Western blotting using anti-KAP1 and anti-β-actin antibodies (B and D). Error bars indicate SEM of 3 technical replicates from each of 2 transfection experiments. Numbers in panels B and D indicate relative amounts of KAP1 protein determined by densitometry after normalization to β-actin.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot

    31) Product Images from "A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue"

    Article Title: A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    Journal: Analytical Biochemistry

    doi: 10.1016/j.ab.2012.03.012

    Comparison of DNA extraction yield between a rapid extraction method and a long extraction method. We adopted a classic phenol-chloroform extraction method combined with proteinase-K digestion overnight as a reference DNA extraction method. The DNA extraction
    Figure Legend Snippet: Comparison of DNA extraction yield between a rapid extraction method and a long extraction method. We adopted a classic phenol-chloroform extraction method combined with proteinase-K digestion overnight as a reference DNA extraction method. The DNA extraction

    Techniques Used: DNA Extraction

    32) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    Rescue of the DNA2 depletion defect by the purified DNA2 protein. DNA substrates bearing different types of ends were incubated with the DNA2-depleted extracts supplemented with either buffer or DNA2 protein. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
    Figure Legend Snippet: Rescue of the DNA2 depletion defect by the purified DNA2 protein. DNA substrates bearing different types of ends were incubated with the DNA2-depleted extracts supplemented with either buffer or DNA2 protein. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.

    Techniques Used: Purification, Incubation, Agarose Gel Electrophoresis

    Depletion of DNA2 inhibits resection of DNA with 5′ adducts. ( A ) DNA substrates bearing different types of ends were incubated at 1.5 ng/μl with the control or DNA2-depleted extracts. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.
    Figure Legend Snippet: Depletion of DNA2 inhibits resection of DNA with 5′ adducts. ( A ) DNA substrates bearing different types of ends were incubated at 1.5 ng/μl with the control or DNA2-depleted extracts. Samples were treated with SDS-EDTA-Proteinase K, and separated on an 1% TAE-agarose gel. The gel was dried and exposed to X-ray film.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    33) Product Images from "Matrix Production, Pigment Synthesis, and Sporulation in a Marine Isolated Strain of Bacillus pumilus"

    Article Title: Matrix Production, Pigment Synthesis, and Sporulation in a Marine Isolated Strain of Bacillus pumilus

    Journal: Marine Drugs

    doi: 10.3390/md13106472

    Determination of amount of biofilm produced by B. pumilus SF214. ( A ) Cells were grown at the indicated temperatures in rich (LB; light gray bars) or sporulation-inducing (DS; dark gray bars) media for 48 h. Cells were then removed, wells were stained and washed, and the OD (570 nm) was determined. ( B ) Biofilm formation at 25 °C in LB medium supplemented with proteinase K or DNase I, as previously indicated [ 14 ].
    Figure Legend Snippet: Determination of amount of biofilm produced by B. pumilus SF214. ( A ) Cells were grown at the indicated temperatures in rich (LB; light gray bars) or sporulation-inducing (DS; dark gray bars) media for 48 h. Cells were then removed, wells were stained and washed, and the OD (570 nm) was determined. ( B ) Biofilm formation at 25 °C in LB medium supplemented with proteinase K or DNase I, as previously indicated [ 14 ].

    Techniques Used: Produced, Staining

    34) Product Images from "Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa"

    Article Title: Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02861-5

    O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: O-antigen-modulated ROS production by extracted LPS and intact bacterial cells ex vivo. Discs of V. vinifera ‘Cabernet Sauvignon’ leaves were treated with 20 μL of a 50 μg/mL solution of purified LPS elicitors (either wzy or wild type LPS) equal to a final amount of 10 μg (based on Kdo content) of LPS, 20 μL of a 10 8 CFU/mL suspension of Xf wild type or wzy cells, or diH 2 0 or 1× PBS-inoculated controls, respectively. a The amplitude of ROS production remained similar for both wild type and wzy LPS, reaching max production at ~4 min, and plateaued starting around 30 min. b Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Total ROS production was not significantly different between discs treated with wild type or wzy extracted LPS. c Intact wzy cells induced a significantly stronger oxidative burst that persisted nearly 20 min longer than leaves inoculated with wild type bacteria (which contained fully polymerized O-antigens). Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. d Total ROS production is reported as area under the curve (AUC) for plot of luminescence intensity over time. Discs treated with wzy cells produced significantly more ROS than discs treated with wild type cells. Graphs represent the mean of 24 replicates per treatment ± standard error of the mean. Treatments with different letters over the bars are statistically different ( P

    Techniques Used: Ex Vivo, Purification, Produced

    In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P
    Figure Legend Snippet: In situ localization of O-antigen-modulated ROS production in the xylem in petioles of inoculated plants. a DAB-mediated tissue printing of petioles at 15 min post-inoculation indicated a strong production of H 2 O 2 specifically in the xylem vessels of grapevines needle inoculated with wzy cells (location of xylem vessels emphasized with dotted outline). Vines inoculated with wild type Xf exhibited H 2 O 2 production predominantly in peripheral collenchyma tissue, with some production in the xylem vessels. Vines inoculated with 1× PBS buffer served as negative controls. b Mean gray value of grayscale-converted DAB-stained images, representing differences in staining intensity. Grayscale intensities vary from 0 to 255; 0 = black, 255 = white, with the values in between representing shades of gray. The mean gray value of DAB-stained images from wzy -inoculated plants is significantly lower than wild type or 1× PBS-inoculated plants, indicating a darker, or more intense stain, and thus higher amounts of H 2 O 2 . Treatments with different letters over the bars are statistically different ( P

    Techniques Used: In Situ, Staining

    35) Product Images from "Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2"

    Article Title: Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    doi: 10.1007/s10162-015-0547-3

    PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.
    Figure Legend Snippet: PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.

    Techniques Used: Polymerase Chain Reaction, Southern Blot, Construct, Recombinant

    36) Product Images from "Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures"

    Article Title: Characterization of Differentiated Quiescent and Nonquiescent Cells in Yeast Stationary-Phase Cultures

    Journal:

    doi: 10.1091/mbc.E07-07-0666

    Venn diagram of transcripts that increased after proteinase K treatment of cell lysates. Transcripts were evaluated that had a ≥1.5-fold increase in abundance after proteinase K treatment of SP culture and Q and NQ cell fraction lysates. The transcripts
    Figure Legend Snippet: Venn diagram of transcripts that increased after proteinase K treatment of cell lysates. Transcripts were evaluated that had a ≥1.5-fold increase in abundance after proteinase K treatment of SP culture and Q and NQ cell fraction lysates. The transcripts

    Techniques Used:

    mRNA abundance in Q and NQ samples treated with or without proteinase K. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) was used to cluster ∼2400 transcripts. Samples include, unseparated SP culture (SP) and Q and NQ
    Figure Legend Snippet: mRNA abundance in Q and NQ samples treated with or without proteinase K. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) was used to cluster ∼2400 transcripts. Samples include, unseparated SP culture (SP) and Q and NQ

    Techniques Used:

    37) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    38) Product Images from "Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8"

    Article Title: Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051696

    U1 snRNP induces type I IFN production. (A) Human PBMCs were stimulated with U1 snRNP (concentration given for total RNA plus protein) complexed to DOTAP (DO), 100 ng/ml LPS, or DOTAP alone, and cytokines were measured. Med, medium control. (B) SDS page of U1 snRNP, 4% stacking, 13.5% separation gel under reducing conditions, and Coomassie blue staining. Lane UF, RNP/Sm antigen, 8.8 μg protein; lane M, SigmaMarker, wide molecular weight range. (C) PBMCs were cultured with 20 μg/ml snRNP, 10 μg/ml poly rI:rC (pIC), snRNP, or poly rI:rC pretreated with RNase A or snRNP pretreated with proteinase K complexed to DOTAP. (D) PBMCs were stimulated with U1 snRNP or U1 snRNA complexed to DOTAP or DOTAP alone. The stimulatory properties of U1 snRNP or snRNA required the presence of an uptake enhancer (not depicted). All experiments show mean ± SEM of one representative out of two or more experiments, each with at least three to six donors.
    Figure Legend Snippet: U1 snRNP induces type I IFN production. (A) Human PBMCs were stimulated with U1 snRNP (concentration given for total RNA plus protein) complexed to DOTAP (DO), 100 ng/ml LPS, or DOTAP alone, and cytokines were measured. Med, medium control. (B) SDS page of U1 snRNP, 4% stacking, 13.5% separation gel under reducing conditions, and Coomassie blue staining. Lane UF, RNP/Sm antigen, 8.8 μg protein; lane M, SigmaMarker, wide molecular weight range. (C) PBMCs were cultured with 20 μg/ml snRNP, 10 μg/ml poly rI:rC (pIC), snRNP, or poly rI:rC pretreated with RNase A or snRNP pretreated with proteinase K complexed to DOTAP. (D) PBMCs were stimulated with U1 snRNP or U1 snRNA complexed to DOTAP or DOTAP alone. The stimulatory properties of U1 snRNP or snRNA required the presence of an uptake enhancer (not depicted). All experiments show mean ± SEM of one representative out of two or more experiments, each with at least three to six donors.

    Techniques Used: Concentration Assay, SDS Page, Staining, Molecular Weight, Cell Culture

    39) Product Images from "GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum"

    Article Title: GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01401-13

    sWGA blot of whole-cell extracts derived from the wild type and tagE5E6 deletion mutant with or without proteinase K treatment for 10, 30, or 60 min. On the left side of the blot the protein sizes are indicated based on the Precision Plus protein dual color standard (Bio-Rad) molecular marker.
    Figure Legend Snippet: sWGA blot of whole-cell extracts derived from the wild type and tagE5E6 deletion mutant with or without proteinase K treatment for 10, 30, or 60 min. On the left side of the blot the protein sizes are indicated based on the Precision Plus protein dual color standard (Bio-Rad) molecular marker.

    Techniques Used: Derivative Assay, Mutagenesis, Marker

    40) Product Images from "Toxoplasma gondii tachyzoite-extract acts as a potent immunomodulator against allergic sensitization and airway inflammation"

    Article Title: Toxoplasma gondii tachyzoite-extract acts as a potent immunomodulator against allergic sensitization and airway inflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15663-4

    In vitro stimulation of bone marrow-derived dendritic cells with heat-inactivated, proteinase K-treated and deglycosylated TLA. Bone marrow-derived dendritic cells (BMDC) were cultured with media only (Med), 5 µg/ml TLA (TLA), heat-inactivated TLA (TLA H), TLA digested with proteinase K (TLA-ProtK), or deglycosylated TLA (TLA D), and LPS (1 µg/ml) for 24 h. Levels of cytokines in culture supernatants were determined by ELISA. All data are representative of at least two independent experiments. Results of Student´s t test: *P
    Figure Legend Snippet: In vitro stimulation of bone marrow-derived dendritic cells with heat-inactivated, proteinase K-treated and deglycosylated TLA. Bone marrow-derived dendritic cells (BMDC) were cultured with media only (Med), 5 µg/ml TLA (TLA), heat-inactivated TLA (TLA H), TLA digested with proteinase K (TLA-ProtK), or deglycosylated TLA (TLA D), and LPS (1 µg/ml) for 24 h. Levels of cytokines in culture supernatants were determined by ELISA. All data are representative of at least two independent experiments. Results of Student´s t test: *P

    Techniques Used: In Vitro, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    41) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    42) Product Images from "Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens"

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02224

    Comparison of DNA extraction protocols in spiked sputum samples. The  M. tuberculosis mc 2 7000  stock suspension was diluted and used to spike negative sputum samples. Box plots with C T  median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ®  method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.
    Figure Legend Snippet: Comparison of DNA extraction protocols in spiked sputum samples. The M. tuberculosis mc 2 7000 stock suspension was diluted and used to spike negative sputum samples. Box plots with C T median, 10th, 25th, 75th, and 90th centiles of 10 replicates. Methods are indicated by colors: brown: Chelex ® method; pink: Guanidium Isothicyanate/Tris-HCl/EDTA + 3 cycles of freeze thawing and boiling; black: Tween 20/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; green: Nonidet P-40/Tris-HCl/EDTA/lysozyme+proteinase K/SDS + warming cycles 56°C/95°C; blue: Triton X-100/Tris-HCl/EDTA; purple: NaOH + boiling and sonication.

    Techniques Used: DNA Extraction, Sonication

    43) Product Images from "PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix"

    Article Title: PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004279

    PINK1 accumulates on the outer membrane and in the matrix upon knockdown of Lon. (A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane–associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown. (C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison. (E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.
    Figure Legend Snippet: PINK1 accumulates on the outer membrane and in the matrix upon knockdown of Lon. (A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane–associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown. (C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison. (E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.

    Techniques Used: Isolation, Expressing, Incubation, Western Blot, Construct, Software

    44) Product Images from "Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2"

    Article Title: Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    doi: 10.1007/s10162-015-0547-3

    PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.
    Figure Legend Snippet: PCR and Southern blot results for five positive ES cell subclones. A PCR genotyping of DNA from the five positive ES cell subclones carrying the Slc44a2 targeting construct (3.3-kb PCR product), subclone 78 that lacked the targeting construct, and +/+ (wild-type) mouse DNA (3.1-kb PCR product) without the construct. B Southern blot showing the presence of the Slc44a2 8.4-kb wild-type and the 6.0-kb recombinant construct in the five positive subclones of clone 29.

    Techniques Used: Polymerase Chain Reaction, Southern Blot, Construct, Recombinant

    45) Product Images from "The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members"

    Article Title: The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members

    Journal: BMC Infectious Diseases

    doi: 10.1186/s12879-016-1651-7

    Six most commonly isolated gram-negative bacteria (from healthy service members) by PCR/ESI-TOF-MS
    Figure Legend Snippet: Six most commonly isolated gram-negative bacteria (from healthy service members) by PCR/ESI-TOF-MS

    Techniques Used: Isolation, Polymerase Chain Reaction, Mass Spectrometry

    Five most commonly detected gram-positive bacteria (from healthy service members) by PCR/ESI-TOF-MS
    Figure Legend Snippet: Five most commonly detected gram-positive bacteria (from healthy service members) by PCR/ESI-TOF-MS

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry

    46) Product Images from "Structural determinants of PINK1 topology and dual subcellular distribution"

    Article Title: Structural determinants of PINK1 topology and dual subcellular distribution

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-11-90

    Subcellular distribution of various PINK1 mutants in Hela cells by fractionation . A) PINK1 MLS-GFP localizes to the mitochondria and is partially sensitive to proteinase K digestion. B) mito-GFP localizes to the mitochondria and is not digested by proteinase K. C) Overexpression of wildtype PINK1-flag displays dual localization for all forms of PINK1. D) Immt-Δ151 PINK1 localizes to the mitochondria and is sensitive to proteinase K digestion. E) mito-Δ151 PINK1 localizes equally in both cytosol and mitochondria, where the mitochondria fraction is not digested by proteinase K. F) Δ90-110 PINK1 mostly localizes to mitochondrial fraction with a small portion of cleaved form redistributed to the cytosolic fraction. G) Δ151 PINK1 localizes mainly in the cytosol with a small pool in the mitochondria. H) Drosophila PINK1-flag (DmPINK1) transfected in Hela cells also distributed to both cytosolic and mitochondrial fractions, similar to human PINK1. ATP5A, the α-subunit of ATP synthase, marks the mitochondria. p38 MAPK marks the cytosol.
    Figure Legend Snippet: Subcellular distribution of various PINK1 mutants in Hela cells by fractionation . A) PINK1 MLS-GFP localizes to the mitochondria and is partially sensitive to proteinase K digestion. B) mito-GFP localizes to the mitochondria and is not digested by proteinase K. C) Overexpression of wildtype PINK1-flag displays dual localization for all forms of PINK1. D) Immt-Δ151 PINK1 localizes to the mitochondria and is sensitive to proteinase K digestion. E) mito-Δ151 PINK1 localizes equally in both cytosol and mitochondria, where the mitochondria fraction is not digested by proteinase K. F) Δ90-110 PINK1 mostly localizes to mitochondrial fraction with a small portion of cleaved form redistributed to the cytosolic fraction. G) Δ151 PINK1 localizes mainly in the cytosol with a small pool in the mitochondria. H) Drosophila PINK1-flag (DmPINK1) transfected in Hela cells also distributed to both cytosolic and mitochondrial fractions, similar to human PINK1. ATP5A, the α-subunit of ATP synthase, marks the mitochondria. p38 MAPK marks the cytosol.

    Techniques Used: Fractionation, Over Expression, Transfection

    47) Product Images from "The Cyclophilin A-CD147 complex promotes bone marrow colonization of B-cell malignancies: implications for therapy"

    Article Title: The Cyclophilin A-CD147 complex promotes bone marrow colonization of B-cell malignancies: implications for therapy

    Journal: Nature medicine

    doi: 10.1038/nm.3867

    In vitro and in vivo migration of MM cells toward BMECs Transwell migration of MM1S-luc cells incubated under different growth conditions: ( a ) in medium alone (Medium), conditioned medium from BMEC-60 cells (BMEC-60-CM) or conditioned medium derived from BMEC-60 cells and treated with proteinase K (BMEC-60-CM + PK); ( b ) in the absence or presence of endothelial cells derived from BM from two different MM persons (PBMEC 1, PBMEC 2); ( c ) in the presence of HS5 cells or PBMEC 1 and PBMSC 1 isolated from same person. Migration data was normalized based on data of Medium alone. Results are means ± SD for assays performed in triplicate. Statistical significance of differences between groups was determined by unpaired Student's t-test. ( d ) Diagram of the three-dimensional poly-ε-caprolactone scaffold xenograft mouse model. Xenogen data ( e ), time course ( f ), and histologic and immunohistochemical analysis ( g ) of MM1S-luc cell growth within non-coated scaffolds or within scaffolds coated with HS5 or BMEC-60 cells. ERG (Ets-related gene): Endothelial cell marker. Bars: Top 100μm, Bottom 20μm. Xenogen data ( h ), time course ( i ), and histologic analysis ( j ) of MM1S-luc cell growth within scaffolds coated with primary BM endothelial cells (PBMEC 1 and PBMEC 2) or primary BM stromal cells (PBMSC 1 and PBMEC 2) isolated from same person with MM. Bars: 50μm. Statistical analysis of tumor burden were done using factorial analysis in SPSS 13.0. The results of two representative experiments of three are shown. (*P
    Figure Legend Snippet: In vitro and in vivo migration of MM cells toward BMECs Transwell migration of MM1S-luc cells incubated under different growth conditions: ( a ) in medium alone (Medium), conditioned medium from BMEC-60 cells (BMEC-60-CM) or conditioned medium derived from BMEC-60 cells and treated with proteinase K (BMEC-60-CM + PK); ( b ) in the absence or presence of endothelial cells derived from BM from two different MM persons (PBMEC 1, PBMEC 2); ( c ) in the presence of HS5 cells or PBMEC 1 and PBMSC 1 isolated from same person. Migration data was normalized based on data of Medium alone. Results are means ± SD for assays performed in triplicate. Statistical significance of differences between groups was determined by unpaired Student's t-test. ( d ) Diagram of the three-dimensional poly-ε-caprolactone scaffold xenograft mouse model. Xenogen data ( e ), time course ( f ), and histologic and immunohistochemical analysis ( g ) of MM1S-luc cell growth within non-coated scaffolds or within scaffolds coated with HS5 or BMEC-60 cells. ERG (Ets-related gene): Endothelial cell marker. Bars: Top 100μm, Bottom 20μm. Xenogen data ( h ), time course ( i ), and histologic analysis ( j ) of MM1S-luc cell growth within scaffolds coated with primary BM endothelial cells (PBMEC 1 and PBMEC 2) or primary BM stromal cells (PBMSC 1 and PBMEC 2) isolated from same person with MM. Bars: 50μm. Statistical analysis of tumor burden were done using factorial analysis in SPSS 13.0. The results of two representative experiments of three are shown. (*P

    Techniques Used: In Vitro, In Vivo, Migration, Incubation, Derivative Assay, Isolation, Immunohistochemistry, Marker

    48) Product Images from "Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols"

    Article Title: Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

    Journal: Journal of Applied Oral Science

    doi: 10.1590/1678-77572016-0046

    DNA quantity, purity, integrity and whether each sample contained amplifiable human DNA obtained from fresh saliva and saliva frozen at different time periods using five different extraction protocols. Protocol 1 (1) used the Oragene™ kit; protocol 2 (2) used the QIAamp® DNA Mini kit; protocol 3 (3) used ammonium acetate, protocol 4 (4) used the InstaGene™ Matrix kit; protocol 5 (5) used the InstaGene™ kit with proteinase K and 1% SDS. Quantity in terms of concentration (ng/µL) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges. (A) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (B) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (C) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (D) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Purity was assessed using spectrophotometry and expressed as a percentage of positive samples, absorbance ratio of A260/280 between 1.6 and 2.0 (black fill) or negative, outside the 1.6 to 2.0 range (white fill); (E) Samples from fresh saliva using each of the 5 DNA extraction protocols; (F) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (G) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (H) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Integrity of DNA, visualized as a percentage of unfragmented (black fill), fragmented (white fill) or undetected (gray fill) DNA, as assessed using electrophoretic analysis; (I) Samples from fresh saliva using each of the 5 DNA extraction protocols; (J) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (K) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (L) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Conventional PCR using primers specific for human DNA was used to amplify exon 3 of the interferon regulatory factor 6 (IRF6) gene in all samples at all time points investigated, and then visualized on an agarose gel; samples were either positive (black fill) or negative (white fill) for the presence of amplified human DNA; (M) Human DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (N) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (O) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (P) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Absolute quantity (µg) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges; (Q) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (R) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (S) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (T) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. For each test at each time point, all protocols were tested overall (p-value
    Figure Legend Snippet: DNA quantity, purity, integrity and whether each sample contained amplifiable human DNA obtained from fresh saliva and saliva frozen at different time periods using five different extraction protocols. Protocol 1 (1) used the Oragene™ kit; protocol 2 (2) used the QIAamp® DNA Mini kit; protocol 3 (3) used ammonium acetate, protocol 4 (4) used the InstaGene™ Matrix kit; protocol 5 (5) used the InstaGene™ kit with proteinase K and 1% SDS. Quantity in terms of concentration (ng/µL) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges. (A) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (B) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (C) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (D) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Purity was assessed using spectrophotometry and expressed as a percentage of positive samples, absorbance ratio of A260/280 between 1.6 and 2.0 (black fill) or negative, outside the 1.6 to 2.0 range (white fill); (E) Samples from fresh saliva using each of the 5 DNA extraction protocols; (F) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (G) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (H) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Integrity of DNA, visualized as a percentage of unfragmented (black fill), fragmented (white fill) or undetected (gray fill) DNA, as assessed using electrophoretic analysis; (I) Samples from fresh saliva using each of the 5 DNA extraction protocols; (J) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (K) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (L) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Conventional PCR using primers specific for human DNA was used to amplify exon 3 of the interferon regulatory factor 6 (IRF6) gene in all samples at all time points investigated, and then visualized on an agarose gel; samples were either positive (black fill) or negative (white fill) for the presence of amplified human DNA; (M) Human DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (N) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (O) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (P) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Absolute quantity (µg) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges; (Q) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (R) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (S) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (T) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. For each test at each time point, all protocols were tested overall (p-value

    Techniques Used: Concentration Assay, Spectrophotometry, DNA Extraction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Significance Assay

    An example of a gel from 5 different extraction protocols when DNA was extracted from fresh saliva or saliva stored for 3, 6 or 12 months investigating whether samples were fragmented. DNA samples from 2 individuals (A, B) were electrophoresed using a 0.8% agarose gel in Tris-acetate (200 mM) with EDTA (50 mM) buffer. Lanes 1 and 25 contain the 100 bp molecular weight standard (M); lanes 2 and 26 contain the positive control (+), 115 ng of human DNA; lanes 3 and 27 contain the negative control (-), 8 µL of ddH2O; lanes 24 and 48 were left blank; all other lanes contain 8 µL of extracted DNA from either volunteer A or B. Protocol 1 (P1) used the Oragene™ kit; protocol 2 (P2) used the QIAamp® DNA Mini kit; protocol 3 (P3) used ammonium acetate, protocol 4 (P4) used the InstaGene™ Matrix kit; protocol 5 (P5) used the InstaGene™ kit with proteinase K and 1% SDS
    Figure Legend Snippet: An example of a gel from 5 different extraction protocols when DNA was extracted from fresh saliva or saliva stored for 3, 6 or 12 months investigating whether samples were fragmented. DNA samples from 2 individuals (A, B) were electrophoresed using a 0.8% agarose gel in Tris-acetate (200 mM) with EDTA (50 mM) buffer. Lanes 1 and 25 contain the 100 bp molecular weight standard (M); lanes 2 and 26 contain the positive control (+), 115 ng of human DNA; lanes 3 and 27 contain the negative control (-), 8 µL of ddH2O; lanes 24 and 48 were left blank; all other lanes contain 8 µL of extracted DNA from either volunteer A or B. Protocol 1 (P1) used the Oragene™ kit; protocol 2 (P2) used the QIAamp® DNA Mini kit; protocol 3 (P3) used ammonium acetate, protocol 4 (P4) used the InstaGene™ Matrix kit; protocol 5 (P5) used the InstaGene™ kit with proteinase K and 1% SDS

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight, Positive Control, Negative Control

    49) Product Images from "Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds"

    Article Title: Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0610-6

    Equine MSC secrete stable, low molecular weight factors that inhibit bacterial growth.  a  Relative growth of  E. coli  ( left panel ) and  S. aureus  ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, heat-inactivated MSC CM ( HI ) or proteinase K-treated CM ( PK ) each diluted 1:2 in LB broth.  b  Relative growth of  E. coli  ( left panel ) and  S. aureus  ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, frozen-thawed MSC CM ( FT ) or lyophilized-reconstituted CM ( Lyoph ) each diluted 1:2 in LB broth.  c  Relative growth of  E. coli  ( left panel ) and  S. aureus  ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, and MSC CM fractioned by size of secreted factors. Different letters indicate statistically significant ( P
    Figure Legend Snippet: Equine MSC secrete stable, low molecular weight factors that inhibit bacterial growth. a Relative growth of E. coli ( left panel ) and S. aureus ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, heat-inactivated MSC CM ( HI ) or proteinase K-treated CM ( PK ) each diluted 1:2 in LB broth. b Relative growth of E. coli ( left panel ) and S. aureus ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, frozen-thawed MSC CM ( FT ) or lyophilized-reconstituted CM ( Lyoph ) each diluted 1:2 in LB broth. c Relative growth of E. coli ( left panel ) and S. aureus ( right panel ) based on absorbance readings at 600 nm. Cultures were grown in DMEM, MSC CM, and MSC CM fractioned by size of secreted factors. Different letters indicate statistically significant ( P

    Techniques Used: Molecular Weight

    50) Product Images from "The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ"

    Article Title: The Probiotic Escherichia coli Strain Nissle 1917 Combats Lambdoid Bacteriophages stx and λ

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00929

    Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p
    Figure Legend Snippet: Phage inactivation studies of EcN and stx -phages. (A) The stx -phage pfus/ml kinetics were analyzed in the presence and absence of EcN in a period of 72 h. Phage titers were determined with the Phage Plaque Assays (PPA) (B) The phage localization PCR with the stx2 primers was performed on the washed bacterial pellet or the sterile filtered supernatant (sn) of EcN or MG1655 after being incubated with stx -phages (EcN stx , MG stx )for 24 h. (C) Heat killed, heat killed and proteinase K (PK) treated or 1% formaldehyde (1% FA) fixed EcN or MG1655 were prepared as described in section Killing of E. coli and their phage inactivation capabilities were compared to the corresponding living E. coli . Bacteria were incubated for 24 h, static before being killed. The heat killed bacteria or the heat killed plus proteinase K treated bacteria were incubated for 24 h with the stx -phages and subsequently the phage titer was determined with the PPA. (D) Heat killed or living SK22D or the commensal control strains SE15 or IAI1 were incubated with the isolated stx -phages for 24 h before the pfus/ml were determined with a PPA. ns, not significant, * p

    Techniques Used: Polymerase Chain Reaction, Incubation, Isolation

    51) Product Images from "Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis"

    Article Title: Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

    Journal: Veterinary Sciences

    doi: 10.3390/vetsci6040088

    Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.
    Figure Legend Snippet: Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.

    Techniques Used: Mass Spectrometry, Labeling, SDS Page, Staining, Silver Staining, Migration

    Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.
    Figure Legend Snippet: Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.

    Techniques Used: Incubation, Binding Assay, Standard Deviation

    52) Product Images from "Extraction and Quantification of Carbon Nanotubes in Biological Matrices with Application to Rat Lung Tissue"

    Article Title: Extraction and Quantification of Carbon Nanotubes in Biological Matrices with Application to Rat Lung Tissue

    Journal: ACS nano

    doi: 10.1021/nn403302s

    Centirfuged rat lung tissue after treatment with the chemical digestion reagents: (a) Solvable, (b) hydroxide, (c) nitric, (d) sulfuric, (e) hydrochloric, (f) hydrofluoric, (g) peroxide, and (h) proteinase K.
    Figure Legend Snippet: Centirfuged rat lung tissue after treatment with the chemical digestion reagents: (a) Solvable, (b) hydroxide, (c) nitric, (d) sulfuric, (e) hydrochloric, (f) hydrofluoric, (g) peroxide, and (h) proteinase K.

    Techniques Used:

    Thermograms for a whole rat lung after treatment with (a) Solvable only and (b) Solvable followed by proteinase K. Data have been cropped to show only the temperatures at which S-CNTs oxidize (i.e., 700–910°C).
    Figure Legend Snippet: Thermograms for a whole rat lung after treatment with (a) Solvable only and (b) Solvable followed by proteinase K. Data have been cropped to show only the temperatures at which S-CNTs oxidize (i.e., 700–910°C).

    Techniques Used:

    53) Product Images from "Susceptibility of Young Sheep to Oral Infection with Bovine Spongiform Encephalopathy Decreases Significantly after Weaning"

    Article Title: Susceptibility of Young Sheep to Oral Infection with Bovine Spongiform Encephalopathy Decreases Significantly after Weaning

    Journal: Journal of Virology

    doi: 10.1128/JVI.01573-12

    Western blot of brain tissue from BSE-challenged sheep brain. Western blot using 6H4 showing proteinase K-treated PrP Sc  in brain tissue from a representative clinically affected sheep challenged with BSE at 6 months of age. Size markers (M), natural scrapie control (1), sheep BSE (2), sheep BSE diluted 1/3 (3), and sheep BSE diluted 1/9 (4). This is a single gel with an irrelevant lane blanked out using Photoshop CS5. No other changes were made.
    Figure Legend Snippet: Western blot of brain tissue from BSE-challenged sheep brain. Western blot using 6H4 showing proteinase K-treated PrP Sc in brain tissue from a representative clinically affected sheep challenged with BSE at 6 months of age. Size markers (M), natural scrapie control (1), sheep BSE (2), sheep BSE diluted 1/3 (3), and sheep BSE diluted 1/9 (4). This is a single gel with an irrelevant lane blanked out using Photoshop CS5. No other changes were made.

    Techniques Used: Western Blot

    54) Product Images from "Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore"

    Article Title: Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore

    Journal: Protein Science : A Publication of the Protein Society

    doi:

    Conformational states of HlyII, HlyII(ΔCT) and αHL examined by limited proteolysis. HlyII, HlyII(ΔCT) and αHL were translated in the presence of rRBCM. The membranes were washed, treated with proteinase K, solubilized and subjected to electrophoresis in a 10% SDS-polyacrylamide gel prior to autoradiography. Lanes 1 – 4 , HlyII; lanes 5 – 8 , HlyII(ΔCT); lanes 9 – 12 , αHL. The final proteinase K concentrations were: lanes 1, 5 , and 9 , 0 μg mL −1 ; lanes 2, 6 , and 10 , 5 μg mL −1 ; lanes 3, 7 , and 11 , 50 μg mL −1 ; lanes 4, 8 , and 12 , 500 μg mL −1 .
    Figure Legend Snippet: Conformational states of HlyII, HlyII(ΔCT) and αHL examined by limited proteolysis. HlyII, HlyII(ΔCT) and αHL were translated in the presence of rRBCM. The membranes were washed, treated with proteinase K, solubilized and subjected to electrophoresis in a 10% SDS-polyacrylamide gel prior to autoradiography. Lanes 1 – 4 , HlyII; lanes 5 – 8 , HlyII(ΔCT); lanes 9 – 12 , αHL. The final proteinase K concentrations were: lanes 1, 5 , and 9 , 0 μg mL −1 ; lanes 2, 6 , and 10 , 5 μg mL −1 ; lanes 3, 7 , and 11 , 50 μg mL −1 ; lanes 4, 8 , and 12 , 500 μg mL −1 .

    Techniques Used: Electrophoresis, Autoradiography

    55) Product Images from "CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing"

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    Journal: mBio

    doi: 10.1128/mBio.01376-18

    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Figure Legend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Techniques Used: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    56) Product Images from "Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection"

    Article Title: Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2019.00460

    Validation of seven selected T. gondii host dependency factors (HDFs). (A) Among the HDFs, seven genes (CBLB, USP17L24, USP19, HDAC7, ULK1, PIM1, and ENPP5) were selected for siRNA gene knockdown assay on Hela cells. Three siRNAs were designed for each gene, and siRNAs with the highest knockdown efficiency were chosen for the further experiments. (B) When specific genes were knocked down with selected siRNA for 24 h, T. gondii was used to infect cells at MOI = 1 for 36 h. Proliferation of T. gondii was significantly inhibited in siRNA treated groups compared with mock and negative control siRNA treated groups. (C,D) FAM-tagged control miRNA inhibitor and mimic transfected Hela cells showed the transfection rate. (E,F) Compared with mock and negative control siRNA treated groups, multiplication of T. gondii was significantly suppressed in specific miRNA inhibitor treated group, but not significantly changed in mimic treated group. (G) Viability of Hela cells in siRNA, inhibitor, and mimic treated groups was not significantly changed. * p
    Figure Legend Snippet: Validation of seven selected T. gondii host dependency factors (HDFs). (A) Among the HDFs, seven genes (CBLB, USP17L24, USP19, HDAC7, ULK1, PIM1, and ENPP5) were selected for siRNA gene knockdown assay on Hela cells. Three siRNAs were designed for each gene, and siRNAs with the highest knockdown efficiency were chosen for the further experiments. (B) When specific genes were knocked down with selected siRNA for 24 h, T. gondii was used to infect cells at MOI = 1 for 36 h. Proliferation of T. gondii was significantly inhibited in siRNA treated groups compared with mock and negative control siRNA treated groups. (C,D) FAM-tagged control miRNA inhibitor and mimic transfected Hela cells showed the transfection rate. (E,F) Compared with mock and negative control siRNA treated groups, multiplication of T. gondii was significantly suppressed in specific miRNA inhibitor treated group, but not significantly changed in mimic treated group. (G) Viability of Hela cells in siRNA, inhibitor, and mimic treated groups was not significantly changed. * p

    Techniques Used: Negative Control, Transfection

    Screening of host dependency factors required by Toxoplasma gondii infection with CRISPR/CAS9-sgRNA library. (A) Screening flowchart. Human foreskin fibroblast (HFF) cells were transduced with lentiviruses at multiplicity of infection 1 (MOI = 1). Cells were subsequently selected using puromycin for 5 days. Transduced cells were infected with T. gondii (MOI = 1) for 10 days. Finally, the transduced cells that survived T. gondii infection were harvested, and the genomic DNA was extracted and subjected to high-throughput sequencing. (B) Venn diagram depicting the number of screened targets from independent biological triplicates: first replicate (red), second replicate (lime), third replicate (blue), and overlapped targets (middle).
    Figure Legend Snippet: Screening of host dependency factors required by Toxoplasma gondii infection with CRISPR/CAS9-sgRNA library. (A) Screening flowchart. Human foreskin fibroblast (HFF) cells were transduced with lentiviruses at multiplicity of infection 1 (MOI = 1). Cells were subsequently selected using puromycin for 5 days. Transduced cells were infected with T. gondii (MOI = 1) for 10 days. Finally, the transduced cells that survived T. gondii infection were harvested, and the genomic DNA was extracted and subjected to high-throughput sequencing. (B) Venn diagram depicting the number of screened targets from independent biological triplicates: first replicate (red), second replicate (lime), third replicate (blue), and overlapped targets (middle).

    Techniques Used: Infection, CRISPR, Transduction, Next-Generation Sequencing

    57) Product Images from "Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress"

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-2-r9

    mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Incubation

    Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
    Figure Legend Snippet: Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T 0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.

    Techniques Used:

    Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
    Figure Legend Snippet: Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T 0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.

    Techniques Used:

    mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2  values for changes in mRNA abundance.
    Figure Legend Snippet: mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log 2 values for changes in mRNA abundance.

    Techniques Used: Isolation, Modification

    58) Product Images from "Pellicle formation in Shewanella oneidensis"

    Article Title: Pellicle formation in Shewanella oneidensis

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-291

    EPS analysis . (A) Effects of proteinase K on pellicle formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent pellicle and supernatant, respectively. Man, gal, and glu represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis.
    Figure Legend Snippet: EPS analysis . (A) Effects of proteinase K on pellicle formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent pellicle and supernatant, respectively. Man, gal, and glu represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis.

    Techniques Used: Concentration Assay, Thin Layer Chromatography, Mutagenesis

    59) Product Images from "Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain"

    Article Title: Modeling Parkinson’s disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1710442114

    Characterization of the aggregates. The pSyn-immunostained aggregates (green) observed in the SN express features characteristic of Lewy bodies (arrowheads) and Lewy neurites (arrows), staining positively for ubiquitin (red) ( A – C ) and thioflavin (green in E ), and being resistant to proteinase K (ProtK) digestion ( D and D ′). Images were captured from an α-SYN + FIB–treated animal at 3 wk post-PFF injection. (Scale bars: A – C , 50 μm; D , 100 μm; D ′, 50 μm; E , 10 μm.)
    Figure Legend Snippet: Characterization of the aggregates. The pSyn-immunostained aggregates (green) observed in the SN express features characteristic of Lewy bodies (arrowheads) and Lewy neurites (arrows), staining positively for ubiquitin (red) ( A – C ) and thioflavin (green in E ), and being resistant to proteinase K (ProtK) digestion ( D and D ′). Images were captured from an α-SYN + FIB–treated animal at 3 wk post-PFF injection. (Scale bars: A – C , 50 μm; D , 100 μm; D ′, 50 μm; E , 10 μm.)

    Techniques Used: Staining, Injection

    60) Product Images from "Using ddPCR to assess the DNA yield of FFPE samples"

    Article Title: Using ddPCR to assess the DNA yield of FFPE samples

    Journal: Biomolecular Detection and Quantification

    doi: 10.1016/j.bdq.2018.10.001

    Schematic workflow for testing various factors in extracting DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. a Commercially available from QIAGEN;  b Included in the QIAamp DNA FFPE Tissue Kit. Abbreviations: PK: Proteinase K; PCE: Phenol-chloroform extraction and ethanol precipitation.
    Figure Legend Snippet: Schematic workflow for testing various factors in extracting DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. a Commercially available from QIAGEN; b Included in the QIAamp DNA FFPE Tissue Kit. Abbreviations: PK: Proteinase K; PCE: Phenol-chloroform extraction and ethanol precipitation.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Ethanol Precipitation

    61) Product Images from "Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress"

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress

    Journal: mSphere

    doi: 10.1128/mSphere.00165-16

    Enriched-variant dispersal. (A) Growth analysis in TSB of the 1457Δ ica , 24c, B5, and A1-35 strains. (B) The 24-h biofilms were treated with enzymes for 2 h and then processed for biofilm formation analysis. (C) Representative images of biofilms following crystal violet staining. Prot K, proteinase K; No Tx, no treatment.
    Figure Legend Snippet: Enriched-variant dispersal. (A) Growth analysis in TSB of the 1457Δ ica , 24c, B5, and A1-35 strains. (B) The 24-h biofilms were treated with enzymes for 2 h and then processed for biofilm formation analysis. (C) Representative images of biofilms following crystal violet staining. Prot K, proteinase K; No Tx, no treatment.

    Techniques Used: Variant Assay, Staining

    62) Product Images from "Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938"

    Article Title: Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01040

    Membrane vesicles of  L. reuteri  DSM 17938 treated with various enzymes. Polymorphic bMVs without an external bilayer ( A , arrow) and pMVs larger than bMVs ( B , arrow) after treatment with DNase I; various and broad distributed bMVs ( C , arrows) and large amount of derived lipid material with irregular shapes and composition obtained by pMVs degradation ( D , arrows) after treatment with Phospholipase C; merged bMVs ( E , arrows) and aggregated pMVs ( F , arrow and arrowhead) in a uniform film treated with Proteinase K; bMVs ( G , arrows) and pMVs ( H , arrows) without treatment (controls).
    Figure Legend Snippet: Membrane vesicles of L. reuteri DSM 17938 treated with various enzymes. Polymorphic bMVs without an external bilayer ( A , arrow) and pMVs larger than bMVs ( B , arrow) after treatment with DNase I; various and broad distributed bMVs ( C , arrows) and large amount of derived lipid material with irregular shapes and composition obtained by pMVs degradation ( D , arrows) after treatment with Phospholipase C; merged bMVs ( E , arrows) and aggregated pMVs ( F , arrow and arrowhead) in a uniform film treated with Proteinase K; bMVs ( G , arrows) and pMVs ( H , arrows) without treatment (controls).

    Techniques Used: Derivative Assay

    63) Product Images from "Whole Genome Amplification of DNA from Laser Capture-Microdissected Tissue for High-Throughput Single Nucleotide Polymorphism and Short Tandem Repeat Genotyping"

    Article Title: Whole Genome Amplification of DNA from Laser Capture-Microdissected Tissue for High-Throughput Single Nucleotide Polymorphism and Short Tandem Repeat Genotyping

    Journal: The American Journal of Pathology

    doi:

    Effect of lysis procedure on a TaqMan SNP-genotyping assay for C/G16996 of the ERBB3 gene. LCM colon cell caps containing 1500 cells (8 caps), 750 cells (12 caps), and 300 cells (8 caps) were lysed by a proteinase K (pk) or an alkaline (alk) lysis protocol. All samples should give a heterozygote call. No calls are highlighted by open circles and miscalls are highlighted by filled circles . Shown on axes are arbitrary fluorescence units.
    Figure Legend Snippet: Effect of lysis procedure on a TaqMan SNP-genotyping assay for C/G16996 of the ERBB3 gene. LCM colon cell caps containing 1500 cells (8 caps), 750 cells (12 caps), and 300 cells (8 caps) were lysed by a proteinase K (pk) or an alkaline (alk) lysis protocol. All samples should give a heterozygote call. No calls are highlighted by open circles and miscalls are highlighted by filled circles . Shown on axes are arbitrary fluorescence units.

    Techniques Used: Lysis, TaqMan SNP Genotyping Assay, Laser Capture Microdissection, Fluorescence

    64) Product Images from "Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus"

    Article Title: Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00185-16

    The TLR2 ligand of  O. tsutsugamushi  is sensitive to proteinase K, H 2 O 2 , and NaOH. (A to D) HEK293 cells transfected with human TLR2 were stimulated with heat-inactivated and crude lysates of  O. tsutsugamushi  pretreated with the indicated compound. IL-8
    Figure Legend Snippet: The TLR2 ligand of O. tsutsugamushi is sensitive to proteinase K, H 2 O 2 , and NaOH. (A to D) HEK293 cells transfected with human TLR2 were stimulated with heat-inactivated and crude lysates of O. tsutsugamushi pretreated with the indicated compound. IL-8

    Techniques Used: Transfection

    Related Articles

    Centrifugation:

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    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
    Article Snippet: The lysate was recovered from the culture flask and heated to 95°C for 10 min to deactivate proteinase K before adding 7.5 U/ml DNase-I (Qiagen) and incubating overnight at 37°C. .. The digested lysate was then mixed 1:1 with 4 M sodium chloride to dissociate GAG-bound peptides, followed by mixing 1:1 with chloroform and centrifugation for 30 min at 4500xg.

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. To test the cytotoxicity of cell culture supernatants infected with M. bovis , mycoplasma cells were killed by a gentamicin treatment (400 μg/ml) for 3 h at 37°C and mycoplasma cells were depleted by 30 min centrifugation at 12,000 ×g (4°C).

    Amplification:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min. .. The spliced and nonspliced RNA was amplified by using the one-step RT-PCR kit (Qiagen) according to the manufacturer's instructions.

    Overlay Assay:

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase
    Article Snippet: Prior to testing in an agar overlay assay, STYM1 extracts were treated with either heat, proteinase K, or passage through a 10-kDa-MWCO filter (Millipore, Burlington, MA). .. The STYM1 extract was heat inactivated at 95°C for 20 min. For proteinase K treatment, 2 μl of proteinase K (20 mg/ml; Qiagen, Germantown, MD) was added to 18 μl of extract, followed by incubation for 1 h at 37°C.

    Cytotoxicity Assay:

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Paragraph title: Co-cultivation of M. bovis With Bovine Lung Cells and Crystal Violet Cell Cytotoxicity Assay ... Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Real-time Polymerase Chain Reaction:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Incubation:

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis
    Article Snippet: The medium containing the filtrate was either untreated (OS7 filtrate), or treated either with 100 μl of proteinase K (Qiagen; > 600 mAU/ml; one mAU releases 1 µmol tyrosine per min from hemoglobin at 37 °C, pH7.5) at 50 °C for 20 min followed by the heat treatment at 100 °C for 5 min (OS7 filtrate + K), or without Qiagen proteinase K at 50 °C for 20 min (OS7 filtrate +50 °C). .. The duplicated samples thus prepared were incubated at 37 °C for 14 days under N2 + CO2 , and the concentrations of Fe2+ , CH4 , and H2 in the samples were determined as described above.

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. ..

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury
    Article Snippet: .. Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C. .. Following digestion, samples were immediately boiled in SDS and processed for western blotting with HDAC1, HDAC2, and ACAA2 (Santa Cruz sc-100847).

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: Standard splicing reactions were carried out in 30% HeLa nuclear extract (Computer Cell Culture Centre, Seneffe, Belgium) in the presence of either DMSO or compound and incubated at 30 °C for 90 min. .. The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min.

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays
    Article Snippet: Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies. .. The LPS patterns of the strains were obtained by silver–staining of sodium dodecyl sulphate polyacrylamide gels [ ] as well as by incubation of strips with an Lp 1 monoclonal antibody from the Dresden panel [ ].

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Article Title: Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase
    Article Snippet: .. The STYM1 extract was heat inactivated at 95°C for 20 min. For proteinase K treatment, 2 μl of proteinase K (20 mg/ml; Qiagen, Germantown, MD) was added to 18 μl of extract, followed by incubation for 1 h at 37°C. ..

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: .. Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. Polynucleotides were removed from DNase I digestion products by using the QIAquick PCR Purification Kit (Qiagen).

    Cell Culture:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: Standard splicing reactions were carried out in 30% HeLa nuclear extract (Computer Cell Culture Centre, Seneffe, Belgium) in the presence of either DMSO or compound and incubated at 30 °C for 90 min. .. The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min.

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. To test the cytotoxicity of cell culture supernatants infected with M. bovis , mycoplasma cells were killed by a gentamicin treatment (400 μg/ml) for 3 h at 37°C and mycoplasma cells were depleted by 30 min centrifugation at 12,000 ×g (4°C).

    BIA-KA:

    Article Title: Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro
    Article Snippet: Proteinase K was obtained from Qiagen (Valencia, CA) and NEB2 buffer was purchased from New England BioLabs (Ipswich, MA). .. BCA Protein Assay Reagent, HPLC grade methanol and chloroform were purchased from Thermo Fisher Scientific, Inc. (Rockford, IL).

    Western Blot:

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury
    Article Snippet: For western blotting, antibodies against HDAC1 (Santa Cruz sc-6298, Abcam 53091), HDAC2 (Santa Cruz sc-7899), HDAC3 (Santa Cruz sc-11417), HDAC8 (Santa Cruz sc-11,405), α/β-tubulin (Cell Signaling 2148) and histone H3 (Cell Signaling 9715) were used. .. Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C.

    Article Title: Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro
    Article Snippet: The chemiluminescent kit, ECL™ Plus Western Blot Detection System and both secondary antibodies, ECL™ Anti-mouse IgG horseradish peroxidase linked F(ab′)2 , and ECL™ Anti-rabbit IgG horseradish peroxidase linked F(ab′)2 were obtained from GE Healthcare (UK Little Chalfont Buckinghamshire). .. Proteinase K was obtained from Qiagen (Valencia, CA) and NEB2 buffer was purchased from New England BioLabs (Ipswich, MA).

    High Performance Liquid Chromatography:

    Article Title: Examination of the Specificity of Tumor Cell Derived Exosomes with Tumor Cells In Vitro
    Article Snippet: Proteinase K was obtained from Qiagen (Valencia, CA) and NEB2 buffer was purchased from New England BioLabs (Ipswich, MA). .. BCA Protein Assay Reagent, HPLC grade methanol and chloroform were purchased from Thermo Fisher Scientific, Inc. (Rockford, IL).

    Silver Staining:

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays
    Article Snippet: Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies. .. The LPS patterns of the strains were obtained by silver–staining of sodium dodecyl sulphate polyacrylamide gels [ ] as well as by incubation of strips with an Lp 1 monoclonal antibody from the Dresden panel [ ].

    Transferring:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

    Infection:

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. To test the cytotoxicity of cell culture supernatants infected with M. bovis , mycoplasma cells were killed by a gentamicin treatment (400 μg/ml) for 3 h at 37°C and mycoplasma cells were depleted by 30 min centrifugation at 12,000 ×g (4°C).

    Generated:

    Article Title: An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis
    Article Snippet: The concentrations of Fe2+ , CH4 , and H2 generated from the cultures were determined as described previously . .. The medium containing the filtrate was either untreated (OS7 filtrate), or treated either with 100 μl of proteinase K (Qiagen; > 600 mAU/ml; one mAU releases 1 µmol tyrosine per min from hemoglobin at 37 °C, pH7.5) at 50 °C for 20 min followed by the heat treatment at 100 °C for 5 min (OS7 filtrate + K), or without Qiagen proteinase K at 50 °C for 20 min (OS7 filtrate +50 °C).

    Confocal Laser Scanning Microscopy:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. ..

    Imaging:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. ..

    Polymerase Chain Reaction:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min. .. PCR products were separated by electrophoresis using 1% agarose gels containing SYBR safe DNA gel stain (Invitrogen).

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. Polynucleotides were removed from DNase I digestion products by using the QIAquick PCR Purification Kit (Qiagen).

    Binding Assay:

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays
    Article Snippet: Immunoblotting To study if the antibody responses, determined in ELISA and IFA with the polyvalent antigens, were directed to the serogroup-specific lipopolysaccharide (LPS) antigen of the Lp 1 outbreak strain [ ], immunoblotting with whole-cell suspensions of this strain was performed as described previously [ ] with detection of IgG and IgM binding on separate strips. .. Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies.

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. Chelex® resin methods in act as chelating groups inactivating nucleases and protecting DNA by binding polyvalent metal ions such as magnesium (Mg2+ ).

    Immunofluorescence:

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays
    Article Snippet: Immunoblotting To study if the antibody responses, determined in ELISA and IFA with the polyvalent antigens, were directed to the serogroup-specific lipopolysaccharide (LPS) antigen of the Lp 1 outbreak strain [ ], immunoblotting with whole-cell suspensions of this strain was performed as described previously [ ] with detection of IgG and IgM binding on separate strips. .. Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies.

    Isolation:

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury
    Article Snippet: Paragraph title: 2.5. Mitochondrial isolation and proteinase K digestion ... Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C.

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress
    Article Snippet: In RNA isolation protocols in which the initial lysis is done with phenol chloroform, protease treatment is not possible. .. Digestion of T0 samples with proteinase K resulted in similar increases of a larger number of transcripts (Figure , lanes 5 and 6), while treatment with Qiagen protease resulted in increases in fewer transcripts (Figure , lanes 7 and 8).

    Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
    Article Snippet: Paragraph title: Isolation of GAGs from cells and tissues ... The lysate was recovered from the culture flask and heated to 95°C for 10 min to deactivate proteinase K before adding 7.5 U/ml DNase-I (Qiagen) and incubating overnight at 37°C.

    Microscopy:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: For microscopy of aggregates, 20 μl of culture was deposited on a glass slide using a P200 pipette tip and imaged using confocal laser scanning microscopy with either a 20× or 63× lens objective. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

    Titration:

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: At different time post-inoculation, mycoplasma titers were determined by CFU titration following one freeze-thaw cycle. .. Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min. .. The spliced and nonspliced RNA was amplified by using the one-step RT-PCR kit (Qiagen) according to the manufacturer's instructions.

    Staining:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min. .. PCR products were separated by electrophoresis using 1% agarose gels containing SYBR safe DNA gel stain (Invitrogen).

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Cell monolayers were washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco) and stained for 60 min with 0.1% crystal violet solution in 10% ethanol. .. Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Activated Clotting Time Assay:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. Chelex® resin methods in act as chelating groups inactivating nucleases and protecting DNA by binding polyvalent metal ions such as magnesium (Mg2+ ).

    Purification:

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. Then supernatant was purified using traditional nucleic acid precipitation: precipitate DNA with 2 volume of ice-cold ethanol and 1/10th volume 3 M sodium acetate, kept at -20°C for 20 min. After centrifugation tubes at 14,000 g for 10 min at 4°C, the pellet was washed with 70% ethanol, air dried, then resuspended in 50 μl TE buffer and used 5 μl in PCR.

    Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
    Article Snippet: The lysate was recovered from the culture flask and heated to 95°C for 10 min to deactivate proteinase K before adding 7.5 U/ml DNase-I (Qiagen) and incubating overnight at 37°C. .. The top (aqueous) layer containing purified GAGs was collected and dialyzed thoroughly against 18.2 MΩ.cm deionized water (MQ).

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C. .. Polynucleotides were removed from DNase I digestion products by using the QIAquick PCR Purification Kit (Qiagen).

    Plasmid Preparation:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: Percent relative aggregation was calculated by taking the difference between the OD600 of the PcdrAB strain and that of its corresponding vector control strain, dividing by the OD600 of the vector control strain, and then multiplying by 100%. .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays
    Article Snippet: From each patient, the serum sample (diluted 1:200) with the highest IgG or IgM antibody level in ELISA was used. .. Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies.

    In Vitro:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: Paragraph title: In Vitro Transcription and Nonradioactive in Vitro Splicing Reaction ... The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min.

    Electrophoresis:

    Article Title: Identification of Small Molecule Inhibitors of Pre-mRNA Splicing *
    Article Snippet: The splicing reaction was followed by a heat inactivation step of 5 min at 95 °C before the samples were subjected to proteinase K (Qiagen) digestion for 30 min at 55 °C and another heat inactivation step at 95 °C for 5 min. .. PCR products were separated by electrophoresis using 1% agarose gels containing SYBR safe DNA gel stain (Invitrogen).

    Concentration Assay:

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing
    Article Snippet: .. For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed. ..

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity
    Article Snippet: To test the influence of extracellular nucleic acids on M. bovis cytotoxicity, mycoplasmas and EBL cells were co-cultivated in DMEM-based medium supplemented with increasing concentration of calf thymus DNA (UltraPure™ Calf Thymus DNA Solution, Invitrogen) and/or yeast tRNA (Invitrogen). .. Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Lysis:

    Article Title: Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress
    Article Snippet: In RNA isolation protocols in which the initial lysis is done with phenol chloroform, protease treatment is not possible. .. Digestion of T0 samples with proteinase K resulted in similar increases of a larger number of transcripts (Figure , lanes 5 and 6), while treatment with Qiagen protease resulted in increases in fewer transcripts (Figure , lanes 7 and 8).

    Article Title: Optimized Lysis-Extraction Method Combined With IS6110-Amplification for Detection of Mycobacterium tuberculosis in Paucibacillary Sputum Specimens
    Article Snippet: .. After vortex mixing, boiling at 100°C for 15 min, then placed in an ultrasonic water bath for 15 min. After centrifugation at 14,000 g for 5 min, the supernatant was used for qPCR. (ii) Guanidium Isothicyanate (GTIC) method: incubation with 200 μl of lysis buffer (10 mM Tris–HCl, 1 mM EDTA, 1 M GTIC, 0.5 M NaCl) for 20 min, combined with 3 cycles of freeze-thawing (-80°C for 5 min and 100°C for 5 min) and boiling at 100°C for 15 min. (iii) Tween 20 method: suspension in 200 μl of lysis buffer [0.45% Tween 20, 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2 ], containing 70 μl of 10 mg/ml Lysozyme and was incubated at 37°C for 1 h. 30 μl of proteinase K (10 mg/ml, Qiagen, Germany) and 2% SDS were added, followed by incubation for 1 h at 56°C to remove PCR inhibitors and heated for 15 min at 100°C to ensure complete mycobacterial lysis. (iv) Non-idet P-40 method: the cell pellet was suspended and subjected to method (iii) but NP-40 instead of Tween 20. (v) Triton method: incubation with 200 μl of lysis buffer [100 mM NaCl, 10 mM Tris–HCl (pH 8.0); 1 mM EDTA and 1% Triton X-100]; was incubated for 20 min at 95°C. (vi) NaOH method: incubation with 200 μl lysis buffer [10 mM Tris–HCl (pH 8.0); 1 mM EDTA, 50 mM sodium hydroxide (NaOH) and 2% SDS] at 95°C for 5 min. A total 1800 μl of pure water was added, then vortex mixed, followed by boiling for 15 min at 100°C. .. After vortex mixing, the tubes were placed in ultrasonic bath for 15 min. For methods (ii) to (vi), after centrifugation at 14,000 g for 5 min, the supernatant was transferred to a new tube.

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    Qiagen proteinase k
    <t>Proteinase</t> K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”
    Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Journal: mBio

    Article Title: CdrA Interactions within the Pseudomonas aeruginosa Biofilm Matrix Safeguard It from Proteolysis and Promote Cellular Packing

    doi: 10.1128/mBio.01376-18

    Figure Lengend Snippet: Proteinase K diminishes the amount of CdrA-dependent aggregation and static biofilm formation. (A) Aggregates of bacteria constitutively expressing GFP were imaged using confocal laser scanning microscopy with proteinase K (PK) treatment or no treatment (NT). Representative images of each strain and condition are shown and were obtained from microscopy of at least three biological replicates. (B) Static biofilm formation of cdrAB overexpression strains (solid lines) and isogenic strains carrying the empty vector control (dashed lines) was measured by crystal violet staining with PK treatment or NT. Data represent the means of results from 3 to 6 replicates, and error bars indicate standard deviations. Scale bars represent 25 μm, and “Δ psl pel algD ” is abbreviated as “Δ EPS .”

    Article Snippet: For proteinase K treatment of aggregates, proteinase K (Qiagen) (final concentration, 5 mg/ml) was added to culture aliquots after 2 h 15 min of growth and incubated for 30 min at room temperature with rocking before imaging by confocal laser scanning microscopy was performed.

    Techniques: Expressing, Confocal Laser Scanning Microscopy, Microscopy, Over Expression, Plasmid Preparation, Staining

    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Journal: Journal of molecular and cellular cardiology

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

    doi: 10.1016/j.yjmcc.2017.12.004

    Figure Lengend Snippet: Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Article Snippet: Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C.

    Techniques: Activity Assay, Western Blot, Staining

    IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.

    Journal: BMC Infectious Diseases

    Article Title: Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays

    doi: 10.1186/s12879-015-0903-2

    Figure Lengend Snippet: IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [ 26 ]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.

    Article Snippet: Blotting was also performed with proteinase K (Qiagen Gmbh, Hilden, Germany) treated cells [ ] from the outbreak strain and from an Lp 1 isolate of subgroup France/Allentown to study LPS cross-reactive antibodies.

    Techniques: Binding Assay, Western Blot, Stripping Membranes, Acrylamide Gel Assay, Incubation

    The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Journal: Frontiers in Microbiology

    Article Title: Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

    doi: 10.3389/fmicb.2019.02753

    Figure Lengend Snippet: The growth-promoting effect of eDNA on M. bovis . Comparative growth of M. bovis under axenic and cell culture conditions (A) . RM16 proliferation was monitored in SP4 medium (SP4), cell culture medium (DMEM), and cell culture medium supplemented with 10 μg/ml calf thymus DNA (DMEMD). Mycoplasma titers (log CFU/ml) were determined by CFU titrations. The cytopathic effect induced by M. bovis upon co-incubation with host cells (B) . EBL cells (10 4 cells) were inoculated with RM16 at an MOI of 2 (RM16) or mock-infected (Mock). After 72 h of co-incubation, monolayers were stained with crystal violet and survival cells were estimated by measuring the optical density at 590 nm (OD 590). When indicated, DMEM-based medium was supplemented with 10 μg/ml calf thymus DNA (eDNA). Calf thymus DNA was subjected to the following enzymatic treatments: RNase A (RNase), Proteinase K (ProtK) and DNase I (DNase) digestion (see section “Materials and Methods”). The asterisk indicates that polynucleotides were removed from DNase I digestion products (DNase*). Infected and mock-infected samples were treated identically. Data are the means of at least three independent assays. Standard deviations are indicated by error bars. p -values were determined by using two-sided independent sample t tests and comparing OD590 values of RM16-infected samples to those of mock-infected samples ( *** p

    Article Snippet: Calf thymus DNA treatment with Proteinase K (Qiagen), DNase I (Invitrogen), and RNase A (Invitrogen) was performed by 1 h incubation in DMEM medium and 15 min heating at 96°C.

    Techniques: Cell Culture, Incubation, Infection, Staining