proteinase k  (New England Biolabs)


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    Name:
    Proteinase K Molecular Biology Grade
    Description:
    Proteinase K Molecular Biology Grade 2 ml
    Catalog Number:
    p8107s
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    83
    Size:
    2 ml
    Category:
    Proteases
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    New England Biolabs proteinase k
    Proteinase K Molecular Biology Grade
    Proteinase K Molecular Biology Grade 2 ml
    https://www.bioz.com/result/proteinase k/product/New England Biolabs
    Average 99 stars, based on 513 article reviews
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    proteinase k - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911"

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    Journal: Scientific Reports

    doi: 10.1038/srep26834

    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Figure Legend Snippet: Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.

    Techniques Used: Derivative Assay, Purification, Quantitative RT-PCR, Centrifugation, Clarification Assay, Fractionation, Size-exclusion Chromatography

    Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).
    Figure Legend Snippet: Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).

    Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR, Size-exclusion Chromatography, Concentration Assay

    2) Product Images from "In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation"

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx049

    Schematic representation of yeast  in vivo  RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+  beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the  GCN4  mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.
    Figure Legend Snippet: Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Techniques Used: In Vivo, RNA Binding Assay, Sequencing, Purification, Mutagenesis, Isolation, Quantitative RT-PCR

    3) Product Images from "Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection"

    Article Title: Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2017.01.016

    The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).
    Figure Legend Snippet: The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).

    Techniques Used: Spectrophotometry, Transmission Electron Microscopy

    4) Product Images from "TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay"

    Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

    Journal: bioRxiv

    doi: 10.1101/299727

    Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.
    Figure Legend Snippet: Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

    Techniques Used: Chromatin Immunoprecipitation, Sonication, Magnetic Beads, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Purification

    5) Product Images from "Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq"

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    Journal: bioRxiv

    doi: 10.1101/792986

    Template-switching reactions with varying concentrations of a 5’-labeled acceptor RNA analyzed by nondenaturing PAGE. Template-switching reactions with varying concentrations of 50-nt acceptor RNA with a 3’-C residue and 50 nM of starter duplex with a 1-nt 3’-G overhang were done for 15 min at 60 °C in reaction medium containing 200 or 450 mM NaCl. After treatment with proteinase K to remove GsI-IIC RT, the products were analyzed by electrophoresis in a nondenaturing 8% polyacrylamide gel. The acceptor RNA (denatured by heating to 82 °C in 1x TE and placed on ice), and a product mimic (labeled acceptor RNA and R2 RNA annealed to a complementary 84-nt oligonucleotide corresponding to the sequence of a full-length cDNA) were loaded in parallel lanes. The gel was run at 4 °C at a constant power of 3 W to maintain the duplex structure and then dried and scanned with a phosphorimager. The labels to the right of the gel indicate the products resulting from the initial template switch (1x) and subsequent end-to-end template switches from the 5’ end of one acceptor to the 3’ end of another (2x, 3x, etc .). Putative product bands were summed and divided by the product and substrate bands to obtain a measure of the fraction product formed, which is indicated under each lane.
    Figure Legend Snippet: Template-switching reactions with varying concentrations of a 5’-labeled acceptor RNA analyzed by nondenaturing PAGE. Template-switching reactions with varying concentrations of 50-nt acceptor RNA with a 3’-C residue and 50 nM of starter duplex with a 1-nt 3’-G overhang were done for 15 min at 60 °C in reaction medium containing 200 or 450 mM NaCl. After treatment with proteinase K to remove GsI-IIC RT, the products were analyzed by electrophoresis in a nondenaturing 8% polyacrylamide gel. The acceptor RNA (denatured by heating to 82 °C in 1x TE and placed on ice), and a product mimic (labeled acceptor RNA and R2 RNA annealed to a complementary 84-nt oligonucleotide corresponding to the sequence of a full-length cDNA) were loaded in parallel lanes. The gel was run at 4 °C at a constant power of 3 W to maintain the duplex structure and then dried and scanned with a phosphorimager. The labels to the right of the gel indicate the products resulting from the initial template switch (1x) and subsequent end-to-end template switches from the 5’ end of one acceptor to the 3’ end of another (2x, 3x, etc .). Putative product bands were summed and divided by the product and substrate bands to obtain a measure of the fraction product formed, which is indicated under each lane.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Sequencing

    6) Product Images from "Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana"

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057283

    PICL and PICC N-termini face the cytoplasm. Immunoblot analysis using GFP antibody. Microsomal preparations were treated with and without Proteinase K. GFP-CXN and CXN-PAGFP were used as controls. In the microsome fraction containing GFP-CXN, GFP is protected from proteinase K treatment, whereas GFP of CXN-PAGFP is susceptible to proteinase K digestion. GFP of GFP-TDF PICL and GFP-TDF PICC are hydrolyzed indicating exposure to Proteinase K. At the given concentration of proteinase K (sufficient to completely hydrolyze GFP in GFP-TDF PICL and GFP-TDF PICC ), a small amount of PAGFP remains undigested (second column of CXN-PAGFP). Microsomal membranes were solubilized by the detergent Triton X-100. Numbers on the left indicate approximate molecular mass in kilodaltons.
    Figure Legend Snippet: PICL and PICC N-termini face the cytoplasm. Immunoblot analysis using GFP antibody. Microsomal preparations were treated with and without Proteinase K. GFP-CXN and CXN-PAGFP were used as controls. In the microsome fraction containing GFP-CXN, GFP is protected from proteinase K treatment, whereas GFP of CXN-PAGFP is susceptible to proteinase K digestion. GFP of GFP-TDF PICL and GFP-TDF PICC are hydrolyzed indicating exposure to Proteinase K. At the given concentration of proteinase K (sufficient to completely hydrolyze GFP in GFP-TDF PICL and GFP-TDF PICC ), a small amount of PAGFP remains undigested (second column of CXN-PAGFP). Microsomal membranes were solubilized by the detergent Triton X-100. Numbers on the left indicate approximate molecular mass in kilodaltons.

    Techniques Used: Concentration Assay

    7) Product Images from "3?-Phosphoadenosine 5?-Phosphosulfate (PAPS) Synthases, Naturally Fragile Enzymes Specifically Stabilized by Nucleotide Binding *"

    Article Title: 3?-Phosphoadenosine 5?-Phosphosulfate (PAPS) Synthases, Naturally Fragile Enzymes Specifically Stabilized by Nucleotide Binding *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.325498

    The V19T mutant of PAPSS2. A , highlighting Thr-29 within the crystal structure PDB code 2OFX of the kinase domain of human PAPSS1. This position of this residue is occupied by valine in PAPSS2. It was also highlighted by random forest analysis. B , unfolding transition for the V19T mutant of PAPSS2. The midpoint of unfolding is lowered from 39.8 to 38.2 °C. C , in presence of APS, however, unfolding is more than rescued. There, the midpoint of the unfolding transition is even shifted to higher temperatures from 45.3 to 47.0 °C indicative of a specific role of threonine at this sequence position. D and E , proteolysis of PAPSS2 by limiting amounts of proteinase K ( PK ). The amount of PK needed to cleave the full-length proteins differs more than 5-fold for wild-type and V19T mutant proteins. F and G , addition of 50 μ m APS stabilized the V19T mutant protein nearly to the wild-type level.
    Figure Legend Snippet: The V19T mutant of PAPSS2. A , highlighting Thr-29 within the crystal structure PDB code 2OFX of the kinase domain of human PAPSS1. This position of this residue is occupied by valine in PAPSS2. It was also highlighted by random forest analysis. B , unfolding transition for the V19T mutant of PAPSS2. The midpoint of unfolding is lowered from 39.8 to 38.2 °C. C , in presence of APS, however, unfolding is more than rescued. There, the midpoint of the unfolding transition is even shifted to higher temperatures from 45.3 to 47.0 °C indicative of a specific role of threonine at this sequence position. D and E , proteolysis of PAPSS2 by limiting amounts of proteinase K ( PK ). The amount of PK needed to cleave the full-length proteins differs more than 5-fold for wild-type and V19T mutant proteins. F and G , addition of 50 μ m APS stabilized the V19T mutant protein nearly to the wild-type level.

    Techniques Used: Mutagenesis, Sequencing

    8) Product Images from "A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice"

    Article Title: A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice

    Journal: eLife

    doi: 10.7554/eLife.53111

    Mono-colonization with curli-sufficient bacteria induce increased αSyn-dependent pathology Germ-free (GF) wild-type (WT) or Thy1-αSyn (ASO) animals were mono-colonized with curli-sufficient  E. coli  (WT) or curli-deficient  E. coli  (Δ csgBAC ). ( A ) Total αSyn in whole brain lysates quantified by ELISA. Quantification of insoluble αSyn fibrils in the ( B ) striatum and ( C ) midbrain by dot blot assay. Midbrain homogenates of ASO animals were ( D ) fractionated with Triton X-100 and SDS-PAGE, and subsequently probed for alpha-synuclein (αSyn) via western blot, or ( E ) left unfractionated and assessed by western blot for phospho-Serine129 αSyn (pS129). ( F–H ) Quantification of western blots by integrated density. Representative images from of mono-colonized ASO mice of the ( I ) midbrain stained for proteinase K-resistant αSyn (green), ( J ) substantia nigra stained for tyrosine hydroxylase (TH, green) and pS129 (red), and ( K ) frontal cortex stained for pS129 (red) and quantified in ( L ). ( M ) Representative images of the myenteric plexus within proximal large intestine of mono-colonized ASO animals stained for PGP9.5 (white) and pS129αSyn (green,), and counts of PGP9.5+ cells bodies per ganglia. ( N ) Proximal large intestine homogenates of mono-colonized ASO animals were assessed by SDS-PAGE and analyzed by western blot for pS129αSyn. Quantification of insoluble αSyn fibrils in the ( O ) duodenum and ( P ) proximal large intestine by dot blot assay. ( Q ) Total striatal dopamine quantified by ELISA. ( R )  TH  expression in the midbrain by qPCR. ( S ) Quantification of TH+ cells in representative sections of the SN. n = 3 ( A–H, L ), n = 4 ( O, P ), n = 7–9 ( Q ), n = 3–4 ( R ), n = 4–5 ( S ). Full-length immunoblots available in associated Source Data file. Points represent individuals, bars represent the mean and standard error. Data analyzed by one-way ANOVA with Tukey post-hoc test for  A-C, Q , or two-tailed  t -test for  F-H, L, O, P, R, S . *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Scale bars = 100 μm. Source data and statistical analysis.
    Figure Legend Snippet: Mono-colonization with curli-sufficient bacteria induce increased αSyn-dependent pathology Germ-free (GF) wild-type (WT) or Thy1-αSyn (ASO) animals were mono-colonized with curli-sufficient E. coli (WT) or curli-deficient E. coli (Δ csgBAC ). ( A ) Total αSyn in whole brain lysates quantified by ELISA. Quantification of insoluble αSyn fibrils in the ( B ) striatum and ( C ) midbrain by dot blot assay. Midbrain homogenates of ASO animals were ( D ) fractionated with Triton X-100 and SDS-PAGE, and subsequently probed for alpha-synuclein (αSyn) via western blot, or ( E ) left unfractionated and assessed by western blot for phospho-Serine129 αSyn (pS129). ( F–H ) Quantification of western blots by integrated density. Representative images from of mono-colonized ASO mice of the ( I ) midbrain stained for proteinase K-resistant αSyn (green), ( J ) substantia nigra stained for tyrosine hydroxylase (TH, green) and pS129 (red), and ( K ) frontal cortex stained for pS129 (red) and quantified in ( L ). ( M ) Representative images of the myenteric plexus within proximal large intestine of mono-colonized ASO animals stained for PGP9.5 (white) and pS129αSyn (green,), and counts of PGP9.5+ cells bodies per ganglia. ( N ) Proximal large intestine homogenates of mono-colonized ASO animals were assessed by SDS-PAGE and analyzed by western blot for pS129αSyn. Quantification of insoluble αSyn fibrils in the ( O ) duodenum and ( P ) proximal large intestine by dot blot assay. ( Q ) Total striatal dopamine quantified by ELISA. ( R ) TH expression in the midbrain by qPCR. ( S ) Quantification of TH+ cells in representative sections of the SN. n = 3 ( A–H, L ), n = 4 ( O, P ), n = 7–9 ( Q ), n = 3–4 ( R ), n = 4–5 ( S ). Full-length immunoblots available in associated Source Data file. Points represent individuals, bars represent the mean and standard error. Data analyzed by one-way ANOVA with Tukey post-hoc test for A-C, Q , or two-tailed t -test for F-H, L, O, P, R, S . *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Scale bars = 100 μm. Source data and statistical analysis.

    Techniques Used: Allele-specific Oligonucleotide, Enzyme-linked Immunosorbent Assay, Dot Blot, SDS Page, Western Blot, Mouse Assay, Staining, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

    9) Product Images from "A Small Wolbachia Protein Directly Represses Phage Lytic Cycle Genes in “Candidatus Liberibacter asiaticus” within Psyllids"

    Article Title: A Small Wolbachia Protein Directly Represses Phage Lytic Cycle Genes in “Candidatus Liberibacter asiaticus” within Psyllids

    Journal: mSphere

    doi: 10.1128/mSphereDirect.00171-17

    Effects of heat and proteinase K treatments on the inhibitory effect of crude aqueous psyllid extracts on fluorimetric GUS activity assays in L . crescens BT-1/pLF057 ( hol :: uidA ) cells. For GUS activity inhibition treatments, 1-ml bacterial culture was incubated overnight with 50 µl psyllid extract, with or without a heat pretreatment (autoclaving at 121 lb/in 2 for 20 min), 2.5 µl proteinase K solution (20 mg ml −1 ), or heat-inactivated proteinase K solution, as indicated. Proteinase K was heat inactivated by incubating at 75°C for 15 min.
    Figure Legend Snippet: Effects of heat and proteinase K treatments on the inhibitory effect of crude aqueous psyllid extracts on fluorimetric GUS activity assays in L . crescens BT-1/pLF057 ( hol :: uidA ) cells. For GUS activity inhibition treatments, 1-ml bacterial culture was incubated overnight with 50 µl psyllid extract, with or without a heat pretreatment (autoclaving at 121 lb/in 2 for 20 min), 2.5 µl proteinase K solution (20 mg ml −1 ), or heat-inactivated proteinase K solution, as indicated. Proteinase K was heat inactivated by incubating at 75°C for 15 min.

    Techniques Used: Activity Assay, Inhibition, Incubation

    10) Product Images from "Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses"

    Article Title: Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03955-8

    Schematic illustration of the ET-B receptor embedded in microsomal membranes and comparison of in-gel-fluorescence and autoradiographic analysis of the synthesized ET-B receptor. Left panel: Shown in red are proteinase K accessible domains that will probably be digested by the enzyme. Proteinase K protected domains are colored in green and blue. Right panel: ET-B was synthesized in the presence of a precharged tRNA harbouring the fluorescence dye Bodipy, coupled to lysine. Bodipy was used to visualize protein bands as alternative to radioactively labeled leucine bands. PNGaseF and ProtK-digestion were performed with the vesicular fraction. Green and blue arrows indicate the corresponding bands to the illustrated domains in the left panel. Black arrows indicate upper bands that might result of incompletely digested domains outside of the microsomal membrane.
    Figure Legend Snippet: Schematic illustration of the ET-B receptor embedded in microsomal membranes and comparison of in-gel-fluorescence and autoradiographic analysis of the synthesized ET-B receptor. Left panel: Shown in red are proteinase K accessible domains that will probably be digested by the enzyme. Proteinase K protected domains are colored in green and blue. Right panel: ET-B was synthesized in the presence of a precharged tRNA harbouring the fluorescence dye Bodipy, coupled to lysine. Bodipy was used to visualize protein bands as alternative to radioactively labeled leucine bands. PNGaseF and ProtK-digestion were performed with the vesicular fraction. Green and blue arrows indicate the corresponding bands to the illustrated domains in the left panel. Black arrows indicate upper bands that might result of incompletely digested domains outside of the microsomal membrane.

    Techniques Used: Fluorescence, Synthesized, Labeling

    11) Product Images from "Intestinal permeability, digestive stability and oral bioavailability of dietary small RNAs"

    Article Title: Intestinal permeability, digestive stability and oral bioavailability of dietary small RNAs

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28207-1

    Circulating form of SCR4 in mice. ( A ) Size exclusion chromatography analysis of mouse sera containing SCR4. Mouse sera rich in SCR4 was loaded to a Sephacryl S-500 column and eluted with 50 mL PBS buffer. Relative levels of SCR4 and miR-16 in selected fractions were quantified by qRT-PCR. The protein abundance in each fraction was measured by spectral absorbance at 280 nm. ( B ) Measurement of resistance of serum SCR4 to proteinase K treatment. Mouse sera rich in SCR4 were mixed with either proteinase K (top panel) or PBS buffer (bottom panel). Aliquots of the reactions were sampled at 5 min, 30 min and 60 min to measure surviving levels of SCR4 or miR-16 by qRT-PCR. For both panel (A,B), a representative experiment from three biological repeats was shown.
    Figure Legend Snippet: Circulating form of SCR4 in mice. ( A ) Size exclusion chromatography analysis of mouse sera containing SCR4. Mouse sera rich in SCR4 was loaded to a Sephacryl S-500 column and eluted with 50 mL PBS buffer. Relative levels of SCR4 and miR-16 in selected fractions were quantified by qRT-PCR. The protein abundance in each fraction was measured by spectral absorbance at 280 nm. ( B ) Measurement of resistance of serum SCR4 to proteinase K treatment. Mouse sera rich in SCR4 were mixed with either proteinase K (top panel) or PBS buffer (bottom panel). Aliquots of the reactions were sampled at 5 min, 30 min and 60 min to measure surviving levels of SCR4 or miR-16 by qRT-PCR. For both panel (A,B), a representative experiment from three biological repeats was shown.

    Techniques Used: Mouse Assay, Size-exclusion Chromatography, Quantitative RT-PCR

    12) Product Images from "Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment"

    Article Title: Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

    Journal: bioRxiv

    doi: 10.1101/684597

    Analysis of the resistant alleles from the tGD( y,w ) arrangement. ( A, B ) Graphs represent the independent generation of specific indel mutations generated in the experiments carried out in Fig. 2 and Fig. S3 when allelic conversion failed at the ( A ) white and ( B ) yellow loci. At both loci, we observe three repeatedly isolated indels that are colored with different shades of red for the white locus (wA, wB, wC) and yellow for the yellow locus (yA, yB, yC), the sequence of which is reported under each graph indicating with dots the missing bases compared to the wild-type sequence, split at the expected cut site. Additional indels recovered more than once are colored in dark grey, and those recovered only once are colored in light gray. ( C-J ) Each panel depicts the number of different indels recovered (y-axis) in ascending order for each F1 female (numbered on x-axis) for which the F2 male progeny was sampled. Bubble size is indicative of the number of flies analyzed for each specific F1 female, reported under the vial number in parenthesis. For F1 females producing only one F2 male with an indel, and therefore only one male was sampled, the dot is represented in magenta.
    Figure Legend Snippet: Analysis of the resistant alleles from the tGD( y,w ) arrangement. ( A, B ) Graphs represent the independent generation of specific indel mutations generated in the experiments carried out in Fig. 2 and Fig. S3 when allelic conversion failed at the ( A ) white and ( B ) yellow loci. At both loci, we observe three repeatedly isolated indels that are colored with different shades of red for the white locus (wA, wB, wC) and yellow for the yellow locus (yA, yB, yC), the sequence of which is reported under each graph indicating with dots the missing bases compared to the wild-type sequence, split at the expected cut site. Additional indels recovered more than once are colored in dark grey, and those recovered only once are colored in light gray. ( C-J ) Each panel depicts the number of different indels recovered (y-axis) in ascending order for each F1 female (numbered on x-axis) for which the F2 male progeny was sampled. Bubble size is indicative of the number of flies analyzed for each specific F1 female, reported under the vial number in parenthesis. For F1 females producing only one F2 male with an indel, and therefore only one male was sampled, the dot is represented in magenta.

    Techniques Used: Generated, Isolation, Western Blot, Sequencing

    13) Product Images from "Nucleosomes impede Cas9 access to DNA in vivo and in vitro"

    Article Title: Nucleosomes impede Cas9 access to DNA in vivo and in vitro

    Journal: eLife

    doi: 10.7554/eLife.12677

    Nucleosomes within chromatinized DNA can block cleavage by Cas9, but a chromatin remodeling factor can restore Cas9 access. ( A ) Schematic of the experimental setup. Supercoiled plasmid containing the 601 sequence inserted into a pBlueScript II SK (+) backbone (pBSIISK+601) was chromatinized by step gradient salt dialysis in the presence of histone octamer. Purified yeast Chd1 (yChd1) remodeling factor was used to test the effect of ATP-dependent remodeling factors on Cas9 access to nucleosomal DNA. ( B ) Quality assessment of the chromatinized plasmid used in this study. Titrated amounts of Micrococcal Nuclease (MNase) were incubated with the chromatinized plasmid, and the resulting pattern of protection by assembled nucleosomes was visualized on a 1.3% agarose gel post-stained with ethidium bromide (EtBr). As a control, the supercoiled plasmid was also incubated with the lowest concentration of MNase. ( C ) A restriction enzyme accessibility assay (REAA) was used to assess the occupancy and position of the nucleosome assembled at the 601 sequence within the chromatinized plasmid. A panel of unique restriction enzyme sites spanning the 601 sequence were incubated with either the supercoiled plasmid, or the chromatinized plasmid. Cleavage was stopped, and protein was removed by incubation with proteinase K followed by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation. (Top) The resulting DNA was then linearized using DraIII, and the level of cleavage by the restriction enzyme panel was visualized on a 1% agarose gel post-stained with EtBr. The label 'P' represents supercoiled plasmid, while 'C' represents chromatinized plasmid. (Bottom right) The location of the restriction sites used are indicated on a diagram of the plasmid. (Bottom left) After quantification of the gel, the percent protection from cleavage experienced in the chromatinized plasmid was plotted versus the location of the cleavage sites on the top strand of the 601 sequence. Experiment 1 refers to the REAA experiment shown in the gel above, while experiment 2 refers to the REAA experiment without remodeler shown in Figure 5F . The grey shading indicates the borders of the 601 sequence, and the grey oval represents the corresponding nucleosome. ( D ) REAA experiment using the frequent cutter, HaeIII, to assess the remodeling activity around the chromatinized plasmid by the purified yChd1 chromatin remodeler. The resulting banding patterns were visualized on a 1.5% agarose gel post-stained with EtBr. Low molecular weight fragments indicate a high degree of HaeIII accessibility, while higher weight bands indicate protection from digestion. ( E ) Diagram showing the location of the restriction enzyme cleavage sites and the PAMs targeted by Cas9/sgRNA in the experiment shown in ( F ) and ( G ). ( F ) An accessibility assay was performed essentially as in C using either restriction enzymes or Cas9/sgRNAs in the presence or absence of the remodeler yChd1. The level of cleavage by the restriction enzyme panel (left) or Cas9/sgRNAs (right) was visualized on a 1.3% agarose gel post-stained with EtBr. A negative control was conducted with an sgRNA that had no sequence complementarity to the plasmid used (non-sense guide). The concentration of yChd1 used was the same as in panel ( D ) ( G ) Quantification of the gels shown in F. Percent protection from cleavage of the chromatinized plasmid in the presence or absence of the chromatin remodeler was calculated relative to the percent cleavage in the corresponding supercoiled plasmid control, and plotted at the location of the restriction enzyme cleavage sites or the center of the PAMs with respect to the 601 dyad. DOI: http://dx.doi.org/10.7554/eLife.12677.012
    Figure Legend Snippet: Nucleosomes within chromatinized DNA can block cleavage by Cas9, but a chromatin remodeling factor can restore Cas9 access. ( A ) Schematic of the experimental setup. Supercoiled plasmid containing the 601 sequence inserted into a pBlueScript II SK (+) backbone (pBSIISK+601) was chromatinized by step gradient salt dialysis in the presence of histone octamer. Purified yeast Chd1 (yChd1) remodeling factor was used to test the effect of ATP-dependent remodeling factors on Cas9 access to nucleosomal DNA. ( B ) Quality assessment of the chromatinized plasmid used in this study. Titrated amounts of Micrococcal Nuclease (MNase) were incubated with the chromatinized plasmid, and the resulting pattern of protection by assembled nucleosomes was visualized on a 1.3% agarose gel post-stained with ethidium bromide (EtBr). As a control, the supercoiled plasmid was also incubated with the lowest concentration of MNase. ( C ) A restriction enzyme accessibility assay (REAA) was used to assess the occupancy and position of the nucleosome assembled at the 601 sequence within the chromatinized plasmid. A panel of unique restriction enzyme sites spanning the 601 sequence were incubated with either the supercoiled plasmid, or the chromatinized plasmid. Cleavage was stopped, and protein was removed by incubation with proteinase K followed by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation. (Top) The resulting DNA was then linearized using DraIII, and the level of cleavage by the restriction enzyme panel was visualized on a 1% agarose gel post-stained with EtBr. The label 'P' represents supercoiled plasmid, while 'C' represents chromatinized plasmid. (Bottom right) The location of the restriction sites used are indicated on a diagram of the plasmid. (Bottom left) After quantification of the gel, the percent protection from cleavage experienced in the chromatinized plasmid was plotted versus the location of the cleavage sites on the top strand of the 601 sequence. Experiment 1 refers to the REAA experiment shown in the gel above, while experiment 2 refers to the REAA experiment without remodeler shown in Figure 5F . The grey shading indicates the borders of the 601 sequence, and the grey oval represents the corresponding nucleosome. ( D ) REAA experiment using the frequent cutter, HaeIII, to assess the remodeling activity around the chromatinized plasmid by the purified yChd1 chromatin remodeler. The resulting banding patterns were visualized on a 1.5% agarose gel post-stained with EtBr. Low molecular weight fragments indicate a high degree of HaeIII accessibility, while higher weight bands indicate protection from digestion. ( E ) Diagram showing the location of the restriction enzyme cleavage sites and the PAMs targeted by Cas9/sgRNA in the experiment shown in ( F ) and ( G ). ( F ) An accessibility assay was performed essentially as in C using either restriction enzymes or Cas9/sgRNAs in the presence or absence of the remodeler yChd1. The level of cleavage by the restriction enzyme panel (left) or Cas9/sgRNAs (right) was visualized on a 1.3% agarose gel post-stained with EtBr. A negative control was conducted with an sgRNA that had no sequence complementarity to the plasmid used (non-sense guide). The concentration of yChd1 used was the same as in panel ( D ) ( G ) Quantification of the gels shown in F. Percent protection from cleavage of the chromatinized plasmid in the presence or absence of the chromatin remodeler was calculated relative to the percent cleavage in the corresponding supercoiled plasmid control, and plotted at the location of the restriction enzyme cleavage sites or the center of the PAMs with respect to the 601 dyad. DOI: http://dx.doi.org/10.7554/eLife.12677.012

    Techniques Used: Blocking Assay, Plasmid Preparation, Sequencing, Purification, Incubation, Agarose Gel Electrophoresis, Staining, Concentration Assay, Ethanol Precipitation, Activity Assay, Molecular Weight, Negative Control

    14) Product Images from "Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity"

    Article Title: Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005618

    Fusion with the redox sensitive BPTI reveals a second translocation step for TM proteins. ( A-D  and  F-L ) Representative images of live  P .  falciparum  parasites expressing the constructs shown schematically above each panel. Hydrophobic regions (SP, signal peptide; TM, transmembrane domain) are in black, the PEXEL motif in yellow. Numbers refer to amino acids (aa). Red boxes labelled C, additional REX2 C-termini. Interrupted yellow box, mutated BPTI (BPTImut). DIC, differential interference contrast. Size bars: 5 μm. ( E ) Schematic for the protease K (PK) protection assay. Left, intact infected RBC with 3 possibilities (I, II, III) for the location of the fusion construct: I, protein is integral to PVM; II, protein is freely accessible in the PV; III, protein is integral to PPM. Middle, after permeabilisation of the erythrocyte plasma membrane (EPM) with tetanolysin the N-terminus of the construct will be digested if it is in the PVM (I), but remains intact in situation II and III. Right, after permeabilisation of the PVM with saponin, the constructs will be digested if it is in the PVM (I) or the PV (II) but if in the PPM (III), an N-terminally truncated fragment will be generated. Red, exported protein; white box, TM; yellow, BPTI with double cysteine bonds; green, GFP. ( F ) Western analysis of a protease protection assay according to ( E ). Digestion is visible after saponin treatment only. As no protected fragment is detectable, the protein is freely accessible in the PV (situation II). The faint bands (asterisk) represent protein degraded down to GFP. REX2-BPTI-GFP was detected using anti-GFP antibodies. Control for PVM integrity was SERA5 (PV resident), for the PPM aldolase (resident in parasite cytoplasm). Release of REX3 (resident of host cell cytosol) demonstrated efficient permeabilisation of the EPM. The marker is indicated in kDa. ( G ) Schematic of the location of REX2 based on the protease protection assay shown in ( F ). Translocation machines are indicated as two black ellipses. Other features are as in ( E ).
    Figure Legend Snippet: Fusion with the redox sensitive BPTI reveals a second translocation step for TM proteins. ( A-D and F-L ) Representative images of live P . falciparum parasites expressing the constructs shown schematically above each panel. Hydrophobic regions (SP, signal peptide; TM, transmembrane domain) are in black, the PEXEL motif in yellow. Numbers refer to amino acids (aa). Red boxes labelled C, additional REX2 C-termini. Interrupted yellow box, mutated BPTI (BPTImut). DIC, differential interference contrast. Size bars: 5 μm. ( E ) Schematic for the protease K (PK) protection assay. Left, intact infected RBC with 3 possibilities (I, II, III) for the location of the fusion construct: I, protein is integral to PVM; II, protein is freely accessible in the PV; III, protein is integral to PPM. Middle, after permeabilisation of the erythrocyte plasma membrane (EPM) with tetanolysin the N-terminus of the construct will be digested if it is in the PVM (I), but remains intact in situation II and III. Right, after permeabilisation of the PVM with saponin, the constructs will be digested if it is in the PVM (I) or the PV (II) but if in the PPM (III), an N-terminally truncated fragment will be generated. Red, exported protein; white box, TM; yellow, BPTI with double cysteine bonds; green, GFP. ( F ) Western analysis of a protease protection assay according to ( E ). Digestion is visible after saponin treatment only. As no protected fragment is detectable, the protein is freely accessible in the PV (situation II). The faint bands (asterisk) represent protein degraded down to GFP. REX2-BPTI-GFP was detected using anti-GFP antibodies. Control for PVM integrity was SERA5 (PV resident), for the PPM aldolase (resident in parasite cytoplasm). Release of REX3 (resident of host cell cytosol) demonstrated efficient permeabilisation of the EPM. The marker is indicated in kDa. ( G ) Schematic of the location of REX2 based on the protease protection assay shown in ( F ). Translocation machines are indicated as two black ellipses. Other features are as in ( E ).

    Techniques Used: Translocation Assay, Expressing, Construct, Infection, Generated, Western Blot, Marker

    15) Product Images from "SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilisIdentification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae"

    Article Title: SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilisIdentification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008296

    SweD and SweC are Type II membrane proteins. ( A ) Fractionation of SweD and SweC analyzed by immunoblot. Lysates from exponentially growing wild-type cells were subject to centrifugation to separate soluble (S100) and membrane-associated (P100) proteins. The membrane fraction was incubated with buffer or buffer supplemented with TritonX-100 and the solubilized proteins (S100) were separated from insoluble material (P100) by a second round of centrifugation. Equivalent amounts of each fraction including the input protoplasts (proto) were separated by SDS-PAGE and analyzed by immunoblot. The membrane protein EzrA and cytoplasmic protein ScpB served as membrane and cytoplasmic controls. ( B ) Protease accessibility analysis of SweD and SweC analyzed by immunoblot. Protoplasts of strain BDR776 were treated with buffer or buffer supplemented with Proteinase K in the absence or presence of sodium-lauroyl-sarcosinate (Sarcosyl). Reactions were resolved by SDS-PAGE and analyzed by immunoblot. SweD and SweC were inaccessible to cleavage by Proteinase K. Degradation of the extracellular domain of SpoIVFA (FA) served as a protease accessible control. The membrane protein EzrA and the cytoplasmic protein FtsE served as protease inaccessible controls. The immunoblots shown are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.
    Figure Legend Snippet: SweD and SweC are Type II membrane proteins. ( A ) Fractionation of SweD and SweC analyzed by immunoblot. Lysates from exponentially growing wild-type cells were subject to centrifugation to separate soluble (S100) and membrane-associated (P100) proteins. The membrane fraction was incubated with buffer or buffer supplemented with TritonX-100 and the solubilized proteins (S100) were separated from insoluble material (P100) by a second round of centrifugation. Equivalent amounts of each fraction including the input protoplasts (proto) were separated by SDS-PAGE and analyzed by immunoblot. The membrane protein EzrA and cytoplasmic protein ScpB served as membrane and cytoplasmic controls. ( B ) Protease accessibility analysis of SweD and SweC analyzed by immunoblot. Protoplasts of strain BDR776 were treated with buffer or buffer supplemented with Proteinase K in the absence or presence of sodium-lauroyl-sarcosinate (Sarcosyl). Reactions were resolved by SDS-PAGE and analyzed by immunoblot. SweD and SweC were inaccessible to cleavage by Proteinase K. Degradation of the extracellular domain of SpoIVFA (FA) served as a protease accessible control. The membrane protein EzrA and the cytoplasmic protein FtsE served as protease inaccessible controls. The immunoblots shown are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.

    Techniques Used: Fractionation, Centrifugation, Incubation, SDS Page, Western Blot, Molecular Weight

    16) Product Images from "Structural insights into assembly and function of the RSC chromatin remodeling complex"

    Article Title: Structural insights into assembly and function of the RSC chromatin remodeling complex

    Journal: bioRxiv

    doi: 10.1101/2020.03.24.006361

    Remodeling assays show that a basic region in Sth1 enhances remodeling. a , DNA substrate used for the restriction enzyme accessibility assays: A 217 bp DNA fragment with the MfeI and PmlI restriction sites used in the assay highlighted. b , Remodeling assays were started with the addition of ATP. Aliquots were removed at 0, 5, 15, 30 and 45 minutes and incubated with Proteinase K to stop the reaction. Deproteinated samples were separated on a 10% TGX gel, stained with SYBR gold, and the uncleaved DNA band (*) was quantified by densitometry.
    Figure Legend Snippet: Remodeling assays show that a basic region in Sth1 enhances remodeling. a , DNA substrate used for the restriction enzyme accessibility assays: A 217 bp DNA fragment with the MfeI and PmlI restriction sites used in the assay highlighted. b , Remodeling assays were started with the addition of ATP. Aliquots were removed at 0, 5, 15, 30 and 45 minutes and incubated with Proteinase K to stop the reaction. Deproteinated samples were separated on a 10% TGX gel, stained with SYBR gold, and the uncleaved DNA band (*) was quantified by densitometry.

    Techniques Used: Incubation, Staining

    17) Product Images from "Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice"

    Article Title: Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188989

    Conformational changes measured by in vitro conversion reactions. (A) In vitro conversion reactions have been performed with radiolabeled wild-type and PrP C (TetraH > G) purified from RK13 cells and PrP Sc purified from brains of RML-infected Tga20 mice as described [ 4 ]. Samples were analyzed by SDS-PAGE-fluorography, and relative conversion efficiencies (CVE) were calculated from band intensities before and after digestion with proteinase K using the formula CVE [%] = [I° +PK / (I° -PK *10)]*100. PrP C with substituted OR histidines (PrP C (TetraH > G)) is only half as efficient in converting to the misfolded, PK-resistant conformer than wt PrP C . Mean values ± standard error (SEM) were determined from 11 independent experiments for each PrP C type. P-values (p (two sided) = 0.07, p (one sided) = 0.036) were obtained by T-Test calculation. ( B ) Control reactions performed in the absence of PrP Sc seed.
    Figure Legend Snippet: Conformational changes measured by in vitro conversion reactions. (A) In vitro conversion reactions have been performed with radiolabeled wild-type and PrP C (TetraH > G) purified from RK13 cells and PrP Sc purified from brains of RML-infected Tga20 mice as described [ 4 ]. Samples were analyzed by SDS-PAGE-fluorography, and relative conversion efficiencies (CVE) were calculated from band intensities before and after digestion with proteinase K using the formula CVE [%] = [I° +PK / (I° -PK *10)]*100. PrP C with substituted OR histidines (PrP C (TetraH > G)) is only half as efficient in converting to the misfolded, PK-resistant conformer than wt PrP C . Mean values ± standard error (SEM) were determined from 11 independent experiments for each PrP C type. P-values (p (two sided) = 0.07, p (one sided) = 0.036) were obtained by T-Test calculation. ( B ) Control reactions performed in the absence of PrP Sc seed.

    Techniques Used: In Vitro, Purification, Infection, Mouse Assay, SDS Page

    Western blot analysis of brain lysates from RML-infected wt and transgenic mice for total PrP C and PrP Sc using monoclonal antibody 4H11. ( A ) Brain homogenates from terminally ill wt and PrP(TetraH > G) line 34 mice were either left untreated (- PK) or subjected to digestion with proteinase K (+ PK). Blots were reprobed for β-actin to control for equal loading. Bands corresponding to total PrP are marked on the left. Irrelevant lanes have been excised at two positions. Molecular weight standards are given on the right (in kDa). ( B ) Corresponding immunoblot analysis of brain homogenates extracted from RML-infected PrP(H95G) mice from the three different lines 11, 13, and 4, respectively, and corresponding wt control (lanes 1 and 2) before (-) and after (+) treatment with PK. Molecular weight standards are given on the right (in kDa).
    Figure Legend Snippet: Western blot analysis of brain lysates from RML-infected wt and transgenic mice for total PrP C and PrP Sc using monoclonal antibody 4H11. ( A ) Brain homogenates from terminally ill wt and PrP(TetraH > G) line 34 mice were either left untreated (- PK) or subjected to digestion with proteinase K (+ PK). Blots were reprobed for β-actin to control for equal loading. Bands corresponding to total PrP are marked on the left. Irrelevant lanes have been excised at two positions. Molecular weight standards are given on the right (in kDa). ( B ) Corresponding immunoblot analysis of brain homogenates extracted from RML-infected PrP(H95G) mice from the three different lines 11, 13, and 4, respectively, and corresponding wt control (lanes 1 and 2) before (-) and after (+) treatment with PK. Molecular weight standards are given on the right (in kDa).

    Techniques Used: Western Blot, Infection, Transgenic Assay, Mouse Assay, Molecular Weight

    18) Product Images from "Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection"

    Article Title: Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection

    Journal: mSphere

    doi: 10.1128/mSphere.00341-18

    Conditioned medium from RSV-infected CF AECs enhances S. aureus biofilm growth. Conditioned medium containing apical secretions from polarized CF AECs was collected from control or RSV-infected cultures. GFP-expressing S. aureus USA100 was inoculated into CM and cultured in glass-bottom MatTek dishes for fluorescence microscopy. Microscopy was performed on a Nikon-Ti wide-field fluorescence microscope. Representative three-dimensional reconstructions of z-stacks obtained for S. aureus biofilm growth in control (A) and RSV (B) CM. Biomass quantification for control and RSV CM (C) was performed in Nikon Elements. Images and biomass quantification are representative of n = 3 individual experiments, with 5 random fields imaged per sample. Total protein content in CM and RSV CM (D). Biofilm growth in untreated control CM and RSV CM and CM pretreated with proteinase K (E) or subjected to 3-kDa centrifugal filtration (F). Biofilm assays were performed in microtiter plates, with a minimum of n = 3 biological replicates and 5 technical replicates per condition, per assay, with crystal violet absorbance quantified at 550 nm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: Conditioned medium from RSV-infected CF AECs enhances S. aureus biofilm growth. Conditioned medium containing apical secretions from polarized CF AECs was collected from control or RSV-infected cultures. GFP-expressing S. aureus USA100 was inoculated into CM and cultured in glass-bottom MatTek dishes for fluorescence microscopy. Microscopy was performed on a Nikon-Ti wide-field fluorescence microscope. Representative three-dimensional reconstructions of z-stacks obtained for S. aureus biofilm growth in control (A) and RSV (B) CM. Biomass quantification for control and RSV CM (C) was performed in Nikon Elements. Images and biomass quantification are representative of n = 3 individual experiments, with 5 random fields imaged per sample. Total protein content in CM and RSV CM (D). Biofilm growth in untreated control CM and RSV CM and CM pretreated with proteinase K (E) or subjected to 3-kDa centrifugal filtration (F). Biofilm assays were performed in microtiter plates, with a minimum of n = 3 biological replicates and 5 technical replicates per condition, per assay, with crystal violet absorbance quantified at 550 nm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: Infection, Expressing, Cell Culture, Fluorescence, Microscopy, Filtration

    19) Product Images from "Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob"

    Article Title: Characterization of an Adenovirus Vector Containing a Heterologous Peptide Epitope in the HI Loop of the Fiber Knob

    Journal: Journal of Virology

    doi:

    Accessibility of the FLAG peptide in the context of intact Ad5F HI FLAG virions. Virions of Ad5F HI FLAG purified on a CsCl gradient were dialyzed, immunoprecipitated with anti-FLAG M2-affinity gel as described in Materials and Methods, and eluted from the gel with free FLAG peptide. Recombinant adenovirus vector Ad5CMVLuc containing unmodified fiber was used as a negative control for binding. Aliquots of all the fractions collected throughout the purification procedure were treated with DNase I to digest traces of the cellular DNA and then treated with SDS, EDTA, and proteinase K to release adenovirus DNA from the virions. The samples obtained were analyzed on a 0.8% agarose gel, and DNA was detected by ethidium bromide staining. Lanes 1 through 3, AdCMVLuc in the supernatant containing unbound material, buffer wash, and FLAG-eluate, respectively; lanes 4 through 6, Ad5F HI FLAG in the supernatant, buffer wash, and FLAG-eluate, respectively; lanes M, DNA molecular weight standards (the bands corresponding to marker fragments ranging from 3 to 12 kb are seen on the gel).
    Figure Legend Snippet: Accessibility of the FLAG peptide in the context of intact Ad5F HI FLAG virions. Virions of Ad5F HI FLAG purified on a CsCl gradient were dialyzed, immunoprecipitated with anti-FLAG M2-affinity gel as described in Materials and Methods, and eluted from the gel with free FLAG peptide. Recombinant adenovirus vector Ad5CMVLuc containing unmodified fiber was used as a negative control for binding. Aliquots of all the fractions collected throughout the purification procedure were treated with DNase I to digest traces of the cellular DNA and then treated with SDS, EDTA, and proteinase K to release adenovirus DNA from the virions. The samples obtained were analyzed on a 0.8% agarose gel, and DNA was detected by ethidium bromide staining. Lanes 1 through 3, AdCMVLuc in the supernatant containing unbound material, buffer wash, and FLAG-eluate, respectively; lanes 4 through 6, Ad5F HI FLAG in the supernatant, buffer wash, and FLAG-eluate, respectively; lanes M, DNA molecular weight standards (the bands corresponding to marker fragments ranging from 3 to 12 kb are seen on the gel).

    Techniques Used: Purification, Immunoprecipitation, Recombinant, Plasmid Preparation, Negative Control, Binding Assay, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

    20) Product Images from "Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection"

    Article Title: Staphylococcus aureus Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection

    Journal: mSphere

    doi: 10.1128/mSphere.00341-18

    Conditioned medium from RSV-infected CF AECs enhances S. aureus biofilm growth. Conditioned medium containing apical secretions from polarized CF AECs was collected from control or RSV-infected cultures. GFP-expressing S. aureus USA100 was inoculated into CM and cultured in glass-bottom MatTek dishes for fluorescence microscopy. Microscopy was performed on a Nikon-Ti wide-field fluorescence microscope. Representative three-dimensional reconstructions of z-stacks obtained for S. aureus biofilm growth in control (A) and RSV (B) CM. Biomass quantification for control and RSV CM (C) was performed in Nikon Elements. Images and biomass quantification are representative of n = 3 individual experiments, with 5 random fields imaged per sample. Total protein content in CM and RSV CM (D). Biofilm growth in untreated control CM and RSV CM and CM pretreated with proteinase K (E) or subjected to 3-kDa centrifugal filtration (F). Biofilm assays were performed in microtiter plates, with a minimum of n = 3 biological replicates and 5 technical replicates per condition, per assay, with crystal violet absorbance quantified at 550 nm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: Conditioned medium from RSV-infected CF AECs enhances S. aureus biofilm growth. Conditioned medium containing apical secretions from polarized CF AECs was collected from control or RSV-infected cultures. GFP-expressing S. aureus USA100 was inoculated into CM and cultured in glass-bottom MatTek dishes for fluorescence microscopy. Microscopy was performed on a Nikon-Ti wide-field fluorescence microscope. Representative three-dimensional reconstructions of z-stacks obtained for S. aureus biofilm growth in control (A) and RSV (B) CM. Biomass quantification for control and RSV CM (C) was performed in Nikon Elements. Images and biomass quantification are representative of n = 3 individual experiments, with 5 random fields imaged per sample. Total protein content in CM and RSV CM (D). Biofilm growth in untreated control CM and RSV CM and CM pretreated with proteinase K (E) or subjected to 3-kDa centrifugal filtration (F). Biofilm assays were performed in microtiter plates, with a minimum of n = 3 biological replicates and 5 technical replicates per condition, per assay, with crystal violet absorbance quantified at 550 nm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: Infection, Expressing, Cell Culture, Fluorescence, Microscopy, Filtration

    21) Product Images from "Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes"

    Article Title: Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211525

    Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30–2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30–2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30–2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T . brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA [ 27 ]. Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods ). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.
    Figure Legend Snippet: Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30–2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30–2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30–2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T . brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA [ 27 ]. Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods ). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.

    Techniques Used: Recombinant, Purification, Staining, Concentration Assay, Incubation

    22) Product Images from "Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies"

    Article Title: Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3625

    Workflows for expansion microscopy with protein retention. Three basic sample processing workflows were explored in this paper. Top , samples are chemically fixed and stained with antibodies, using conventional immunostaining protocols, before AcX treatment at room temperature and subsequent ExM processing (gelation, proteinase K treatment, and expansion in water). Middle , samples expressing fluorescent proteins (FPs) are chemically fixed (and optionally permeabilized) before AcX treatment, and subsequent ExM processing. Bottom , samples treated with AcX, followed by gelation, are then processed with a gentle homogenization procedure (e.g., alkaline hydrolysis and denaturation, or digestion with LysC), and finally antibody staining in the expanded state.
    Figure Legend Snippet: Workflows for expansion microscopy with protein retention. Three basic sample processing workflows were explored in this paper. Top , samples are chemically fixed and stained with antibodies, using conventional immunostaining protocols, before AcX treatment at room temperature and subsequent ExM processing (gelation, proteinase K treatment, and expansion in water). Middle , samples expressing fluorescent proteins (FPs) are chemically fixed (and optionally permeabilized) before AcX treatment, and subsequent ExM processing. Bottom , samples treated with AcX, followed by gelation, are then processed with a gentle homogenization procedure (e.g., alkaline hydrolysis and denaturation, or digestion with LysC), and finally antibody staining in the expanded state.

    Techniques Used: Microscopy, Staining, Immunostaining, Expressing, Homogenization

    23) Product Images from "Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing"

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz1124

    Viral photoactivatable-ribonucleoside-enhanced crosslinking (vPAR-CL) method allows for rapid and high-throughput detection of viral RNA–capsid interactions within virus particles. 4-Thiouridine (4SU) is supplemented to S2 cells during Flock House virus (FHV) infection. After incubation, purified viruses are irradiated with 365nm UV to induce RNA-protein crosslinks. After proteinase K digestion, crosslinked RNAs are purified and used as templates for RNAseq using ClickSeq. By mapping RNAseq reads to the viral genome, the crosslinked sites are characterized by elevated U to C transition rates.
    Figure Legend Snippet: Viral photoactivatable-ribonucleoside-enhanced crosslinking (vPAR-CL) method allows for rapid and high-throughput detection of viral RNA–capsid interactions within virus particles. 4-Thiouridine (4SU) is supplemented to S2 cells during Flock House virus (FHV) infection. After incubation, purified viruses are irradiated with 365nm UV to induce RNA-protein crosslinks. After proteinase K digestion, crosslinked RNAs are purified and used as templates for RNAseq using ClickSeq. By mapping RNAseq reads to the viral genome, the crosslinked sites are characterized by elevated U to C transition rates.

    Techniques Used: High Throughput Screening Assay, Infection, Incubation, Purification, Irradiation

    24) Product Images from "Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells"

    Article Title: Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells

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

    doi:

    Isolation of single fluorescent cells from bone marrow by micromanipulation and preparation of DNA after proteinase K (PK) treatment. After PK inactivation, the double-stranded DNA was digested with Mse I, leaving a TA overhang for adapter annealing and subsequent ligation. After primary amplification, 1/100 of the PCR products was reamplified in the presence of bio-UTP, and 2 μg were used for hybridization.
    Figure Legend Snippet: Isolation of single fluorescent cells from bone marrow by micromanipulation and preparation of DNA after proteinase K (PK) treatment. After PK inactivation, the double-stranded DNA was digested with Mse I, leaving a TA overhang for adapter annealing and subsequent ligation. After primary amplification, 1/100 of the PCR products was reamplified in the presence of bio-UTP, and 2 μg were used for hybridization.

    Techniques Used: Isolation, Micromanipulation, Ligation, Amplification, Polymerase Chain Reaction, Hybridization

    25) Product Images from "Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia"

    Article Title: Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01640-13

    Functional complementation of  E. coli  and  S. enterica  mutants in  waa  genes with  Francisella tularensis  genes. (A)  E. coli  mutants in  waaB ,  waaI , and  waaJ  from the Keio collection were complemented with a plasmid carrying either  FTT1236  (pSL129) or  FTT1237  (pSL30). Proteinase K-treated whole-cell lysates were prepared for each strain, separated by PAGE, and visualized by silver staining. *, upward mobility shift of lipid A-core molecules in each  E. coli  strain. (B) Whole-cell lysates (proteinase K treated) of  S. enterica  SL1344, the  S. enterica waaL  mutant, and the  S. enterica waaL  mutant complemented with  FTT1238c  (pSL149) were separated by PAGE, and O-acylated species were visualized by silver staining.
    Figure Legend Snippet: Functional complementation of E. coli and S. enterica mutants in waa genes with Francisella tularensis genes. (A) E. coli mutants in waaB , waaI , and waaJ from the Keio collection were complemented with a plasmid carrying either FTT1236 (pSL129) or FTT1237 (pSL30). Proteinase K-treated whole-cell lysates were prepared for each strain, separated by PAGE, and visualized by silver staining. *, upward mobility shift of lipid A-core molecules in each E. coli strain. (B) Whole-cell lysates (proteinase K treated) of S. enterica SL1344, the S. enterica waaL mutant, and the S. enterica waaL mutant complemented with FTT1238c (pSL149) were separated by PAGE, and O-acylated species were visualized by silver staining.

    Techniques Used: Functional Assay, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Silver Staining, Mobility Shift, Mutagenesis

    26) Product Images from "A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms"

    Article Title: A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

    Journal: Journal of Virology

    doi: 10.1128/JVI.03014-14

    Protease susceptibility assays with H5 and h8A8. H5 was visualized in Western blotting with MAb 7H10, an antibody targeting the N terminus of H5. (A) h8A8 was able to protect H5 (CDC669) from low-pH-mediated degradation by protease K. 3H12 is an HI antibody
    Figure Legend Snippet: Protease susceptibility assays with H5 and h8A8. H5 was visualized in Western blotting with MAb 7H10, an antibody targeting the N terminus of H5. (A) h8A8 was able to protect H5 (CDC669) from low-pH-mediated degradation by protease K. 3H12 is an HI antibody

    Techniques Used: Western Blot

    27) Product Images from "Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming"

    Article Title: Mitochondrial assembly of the NLRP3 inflammasome complex is initiated at priming

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701723

    NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.
    Figure Legend Snippet: NLRP3 and caspase-1 independently associate with the mitochondria at priming. (A, B) J774A.1 cells were unstimulated, LPS-primed, or LPS-primed followed by stimulation with nigericin (A) or primed with Pam3CSK4 or HMW poly(I:C) (B) . Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (C) J774A.1 cells were LPS-primed for the indicated time in the presence of 10 μM cycloheximide. Mitochondrial and cytosolic fractions were then immunoblotted as indicated. (D) J774A.1 cells were unstimulated or LPS-primed followed by stimulation with nigericin as indicated. Mitochondrial and cytosolic fractions were isolated and mitochondria treated with proteinase K in the presence or absence of Triton X-100. Cytosolic and mitochondrial fractions were then immunoblotted as shown. (E-G) WT, Nlrp3 −/− , Casp1 −/− , and Asc −/− BMM were unstimulated or LPS-primed and stimulated with nigericin as indicated. Mitochondrial and cytosolic fractions were subjected to immunoblot. Data shown are representative of three (A, E, G) or two (B-D, F) independent experiments.

    Techniques Used: Isolation

    28) Product Images from "Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix ▿"

    Article Title: Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00400-09

    Micrographs of biofilm samples treated with different extractants and stained with PI and SYTO-9. Biofilm samples without treatment (A) or treated with CER (B), EDTA (C), SDS (D), NaOH (E), N -glycanase (F), dispersin B (G), or proteinase K (H) are included.
    Figure Legend Snippet: Micrographs of biofilm samples treated with different extractants and stained with PI and SYTO-9. Biofilm samples without treatment (A) or treated with CER (B), EDTA (C), SDS (D), NaOH (E), N -glycanase (F), dispersin B (G), or proteinase K (H) are included.

    Techniques Used: Staining

    29) Product Images from "SaeRS-Dependent Inhibition of Biofilm Formation in Staphylococcus aureus Newman"

    Article Title: SaeRS-Dependent Inhibition of Biofilm Formation in Staphylococcus aureus Newman

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123027

    The biofilm inhibitory factor is a heat stable protein. Stationary phase culture supernatants of strain Newman (wt) and Newman ΔsaeRS were harvested, filter sterilized, and diluted into biofilm medium. Anti-biofilm activity of supernatants was tested against S . aureus strain UAMS-1. ( A ) Proteinase K treatment of culture supernatants. Supernatants from the indicated strains were incubated with proteinase K and Tris buffer, or buffer only (mock prtK), at 42°C for 1 hr and then 95°C for 10 minutes, before adding to biofilm medium. “None” indicates wells containing UAMS-1 but no culture supernatant. Wells labeled “medium” contained sterile biofilm medium. ( B ) Supernatants were incubated at the indicated temperatures for the indicated time periods before adding to wells containing UAMS-1 suspended in biofilm medium.
    Figure Legend Snippet: The biofilm inhibitory factor is a heat stable protein. Stationary phase culture supernatants of strain Newman (wt) and Newman ΔsaeRS were harvested, filter sterilized, and diluted into biofilm medium. Anti-biofilm activity of supernatants was tested against S . aureus strain UAMS-1. ( A ) Proteinase K treatment of culture supernatants. Supernatants from the indicated strains were incubated with proteinase K and Tris buffer, or buffer only (mock prtK), at 42°C for 1 hr and then 95°C for 10 minutes, before adding to biofilm medium. “None” indicates wells containing UAMS-1 but no culture supernatant. Wells labeled “medium” contained sterile biofilm medium. ( B ) Supernatants were incubated at the indicated temperatures for the indicated time periods before adding to wells containing UAMS-1 suspended in biofilm medium.

    Techniques Used: Activity Assay, Incubation, Labeling

    30) Product Images from "A rationally designed peptide enhances homologous recombination in vitro and resistance to DNA damaging agents in vivo"

    Article Title: A rationally designed peptide enhances homologous recombination in vitro and resistance to DNA damaging agents in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq182

    Peptide #3 can enhance both RecA-mediated strand assimilation and three-strand exchange. ( A ) EMSA analysis: formation of RecA–ssDNA filament with or without peptide #3. ΦX 174 was incubated with RecA proteins, peptide #3, or both. The resulting products were separated on an agarose gel and visualized by staining with SYBR-Green II (Invitrogen, USA). ( B ) Time-course experiments of RecA-mediated ATPase assay with or without peptide #3. ATP hydrolysis was initiated by adding 7.5 mM ATP (with 1 nM [γ- 32 P]ATP) at 37°C. At different time points, 0.5 μl aliquots were withdrawn and spotted on a thin layer of chromatography paper to separate [γ- 32 P]ATP from free  32 P-labeled inorganic phosphate. ( C ) The representative gel shows the RecA-mediated three-strand exchange activity with increasing amounts of peptide #3 (0, 3, 10, 50 and 100 µM). The reaction solutions contained an ATP-regeneration system (ATP, phosphocreatine and creatine phosphokinase), Mg(OAc) 2,  RecA proteins, ssDNA (ϕX174), dsDNA (ϕX174RF I, XhoI-treated) and SSB. All reactions were incubated at 37°C. At a determined time, the reactions were stopped by treating with SDS and proteinase K. The samples were analyzed by electrophoresis on an agarose gel using TAE buffer. The substrates and products were visualized by SYBR-Green II (Invitrogen) staining. The relative band intensity was shown above each bar when the intensity of RecA without peptides in 90 min was normalized. See the ‘Materials and Methods’ section for more detailed information. For abbreviations: css, circular single-stranded DNA; lds, linear duplex DNA; jm, joint molecule DNA (D-loop); nc, nicked circular duplex DNA (products).
    Figure Legend Snippet: Peptide #3 can enhance both RecA-mediated strand assimilation and three-strand exchange. ( A ) EMSA analysis: formation of RecA–ssDNA filament with or without peptide #3. ΦX 174 was incubated with RecA proteins, peptide #3, or both. The resulting products were separated on an agarose gel and visualized by staining with SYBR-Green II (Invitrogen, USA). ( B ) Time-course experiments of RecA-mediated ATPase assay with or without peptide #3. ATP hydrolysis was initiated by adding 7.5 mM ATP (with 1 nM [γ- 32 P]ATP) at 37°C. At different time points, 0.5 μl aliquots were withdrawn and spotted on a thin layer of chromatography paper to separate [γ- 32 P]ATP from free 32 P-labeled inorganic phosphate. ( C ) The representative gel shows the RecA-mediated three-strand exchange activity with increasing amounts of peptide #3 (0, 3, 10, 50 and 100 µM). The reaction solutions contained an ATP-regeneration system (ATP, phosphocreatine and creatine phosphokinase), Mg(OAc) 2, RecA proteins, ssDNA (ϕX174), dsDNA (ϕX174RF I, XhoI-treated) and SSB. All reactions were incubated at 37°C. At a determined time, the reactions were stopped by treating with SDS and proteinase K. The samples were analyzed by electrophoresis on an agarose gel using TAE buffer. The substrates and products were visualized by SYBR-Green II (Invitrogen) staining. The relative band intensity was shown above each bar when the intensity of RecA without peptides in 90 min was normalized. See the ‘Materials and Methods’ section for more detailed information. For abbreviations: css, circular single-stranded DNA; lds, linear duplex DNA; jm, joint molecule DNA (D-loop); nc, nicked circular duplex DNA (products).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, ATPase Assay, Thin Layer Chromatography, Labeling, Activity Assay, Electrophoresis

    31) Product Images from "Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)"

    Article Title: Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP)

    Journal: Nature methods

    doi: 10.1038/nmeth.3810

    Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.
    Figure Legend Snippet: Improved identification of RNA binding protein (RBP) targets by enhanced C ross L inking and I mmuno P recipitation followed by high-throughput sequencing (eCLIP-seq) (a) RBP-RNA interactions are stabilized with UV crosslinking, followed by limited RNase I digestion, immunoprecipitation of RBP-RNA complexes with a specific antibody of interest, and stringent washes. After dephosphorylation of RNA fragments, an “inline barcoded” RNA adapter is ligated to the 3′ end. After protein gel electrophoresis and nitrocellulose membrane transfer, a region 75 kDa (~220 nt of RNA) above the protein size is excised and proteinase K treated to isolate RNA. RNA is further prepared into paired-end high-throughput sequencing libraries, where read 1 begins with the inline barcode and read 2 begins with a random-mer sequence (added during the 3′ DNA adapter ligation) followed by sequence corresponding to the 5′ end of the original RNA fragment (which often marks reverse transcriptase termination at the crosslink site (red X)). (b) Bars indicate the number of reads remaining after processing steps. PCR duplicate reads that map to the same genomic position and have the same random-mer as previously considered reads are discarded, leaving only “Usable reads”. (c) Varying numbers of uniquely mapped reads were randomly sampled from RBFOX2 iCLIP and eCLIP experiments and PCR duplicate removal was performed. Points indicate the mean of 100 downsampling experiments (for all, s.e.m. is less than 0.1% of mean value). (d) RBFOX2 read density in reads per million usable (RPM). Shown are iCLIP, two biological replicates for eCLIP with paired size-matched input (SMInput) and IgG-only controls. CLIPper-identified clusters indicated as boxes below, with dark colored boxes indicating binding sites enriched above SMInput.

    Techniques Used: RNA Binding Assay, Next-Generation Sequencing, Immunoprecipitation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Sequencing, Ligation, Polymerase Chain Reaction, Binding Assay

    32) Product Images from "Protein imprinting in polyacrylamide-based gels"

    Article Title: Protein imprinting in polyacrylamide-based gels

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2014.05.079

    Influence of imprinted protein concentration on rebinding. AAm/NIPAm films (20 wt.% monomer and 2 mol.% cross-linker) were imprinted with 0.0083, 0.045, and 0.21 mg mL −1  MBP. After protein removal by digestion in proteinase K, films were incubated in 0.50 mg mL −1  MBP for 5 h. Protein uptake was determined from fluorescence images after rinsing in 10 mM TRIS buffer for 5 min. Each experiment was performed in triplicate. Error bars represent standard deviation. The dotted line has a slope of 1.0.
    Figure Legend Snippet: Influence of imprinted protein concentration on rebinding. AAm/NIPAm films (20 wt.% monomer and 2 mol.% cross-linker) were imprinted with 0.0083, 0.045, and 0.21 mg mL −1 MBP. After protein removal by digestion in proteinase K, films were incubated in 0.50 mg mL −1 MBP for 5 h. Protein uptake was determined from fluorescence images after rinsing in 10 mM TRIS buffer for 5 min. Each experiment was performed in triplicate. Error bars represent standard deviation. The dotted line has a slope of 1.0.

    Techniques Used: Protein Concentration, Incubation, Fluorescence, Standard Deviation

    Protein uptake and elution in imprinted films. (a) AAm/NIPAm films (20 wt.% monomer and 2 mol.% cross-linker) imprinted with 0.21 mg mL −1 MBP were incubated in 10 mM TRIS buffer for 58 h. At each time point films were rinsed for 5 min, imaged, and then incubated in clean buffer. Each experiment was performed in triplicate. Error bars represent standard deviation. (b) After protein elution, films were incubated in proteinase K for 1000 min resulting in almost complete removal of the remaining protein. (c) After protein digestion, films were incubated in 0.50 mg mL −1 MBP solution for 5 h. Error bars represent standard deviation. (d) Following protein rebinding, the films were incubated in 10 mM TRIS buffer to measure protein elution. Error bars represent standard deviation.
    Figure Legend Snippet: Protein uptake and elution in imprinted films. (a) AAm/NIPAm films (20 wt.% monomer and 2 mol.% cross-linker) imprinted with 0.21 mg mL −1 MBP were incubated in 10 mM TRIS buffer for 58 h. At each time point films were rinsed for 5 min, imaged, and then incubated in clean buffer. Each experiment was performed in triplicate. Error bars represent standard deviation. (b) After protein elution, films were incubated in proteinase K for 1000 min resulting in almost complete removal of the remaining protein. (c) After protein digestion, films were incubated in 0.50 mg mL −1 MBP solution for 5 h. Error bars represent standard deviation. (d) Following protein rebinding, the films were incubated in 10 mM TRIS buffer to measure protein elution. Error bars represent standard deviation.

    Techniques Used: Incubation, Standard Deviation

    33) Product Images from "Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells"

    Article Title: Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1523097

    In vitro endothelial cell activation and upregulation of adhesion molecule surface expression are mediated by protein DAMPs derived from irradiated tumor cells. (a) Representative photographs of immunofluorescent adhesion molecule surface staining on HUVECs 4 h after exposure to supernatants of irradiated HCC1937 cells. Surface expression was visualized by immunofluorescence microscopy on native, non-fixed HUVECs. Medium and TNF (50 ng/ml) served as controls. 63x magnification, scale bar 50 µm. (b) Quantitation of ICAM-1 surface expression on HUVECs by fluorometric measurement. HUVECs were treated as in (a) and subjected to native immunofluorescence staining. Staining intensities were quantified by fluorometric measurement, and x-fold expression levels were calculated as the means of fluorescence intensities subtracted by the corresponding isotype controls and normalized to the 0 Gy samples (n = 9 independent experiments). p -values were calculated by unpaired Student’s t -tests with Bonferroni-Holm correction. (c) Biochemical characterization of the molecular entities mediating upregulation of ICAM-1 expression. Supernatants of 20 Gy-irradiated HCC1937 cells were applied to membrane centrifugation (molecular weight cut-off 10 kDa) or proteinase K treatment prior to incubation with HUVECs. ICAM-1 surface expression was measured as in (b) (n = 5–10 independent experiments). Group comparison was performed by unpaired Student’s t -test with Bonferroni-Holm correction. (d) HSP70, HMGB1, and S100A8/A9 were quantified in supernatants of irradiated HCC1937 cells by ELISA. Concentrations were calculated on the basis of standard curves. Means ± SD of 3 (HSP70), 4 (HMGB1), or 5 (S100A8/A9) independent experiments are shown. Group comparison was carried out by two-way ANOVA with Bonferroni-Holm correction.
    Figure Legend Snippet: In vitro endothelial cell activation and upregulation of adhesion molecule surface expression are mediated by protein DAMPs derived from irradiated tumor cells. (a) Representative photographs of immunofluorescent adhesion molecule surface staining on HUVECs 4 h after exposure to supernatants of irradiated HCC1937 cells. Surface expression was visualized by immunofluorescence microscopy on native, non-fixed HUVECs. Medium and TNF (50 ng/ml) served as controls. 63x magnification, scale bar 50 µm. (b) Quantitation of ICAM-1 surface expression on HUVECs by fluorometric measurement. HUVECs were treated as in (a) and subjected to native immunofluorescence staining. Staining intensities were quantified by fluorometric measurement, and x-fold expression levels were calculated as the means of fluorescence intensities subtracted by the corresponding isotype controls and normalized to the 0 Gy samples (n = 9 independent experiments). p -values were calculated by unpaired Student’s t -tests with Bonferroni-Holm correction. (c) Biochemical characterization of the molecular entities mediating upregulation of ICAM-1 expression. Supernatants of 20 Gy-irradiated HCC1937 cells were applied to membrane centrifugation (molecular weight cut-off 10 kDa) or proteinase K treatment prior to incubation with HUVECs. ICAM-1 surface expression was measured as in (b) (n = 5–10 independent experiments). Group comparison was performed by unpaired Student’s t -test with Bonferroni-Holm correction. (d) HSP70, HMGB1, and S100A8/A9 were quantified in supernatants of irradiated HCC1937 cells by ELISA. Concentrations were calculated on the basis of standard curves. Means ± SD of 3 (HSP70), 4 (HMGB1), or 5 (S100A8/A9) independent experiments are shown. Group comparison was carried out by two-way ANOVA with Bonferroni-Holm correction.

    Techniques Used: In Vitro, Activation Assay, Expressing, Derivative Assay, Irradiation, Staining, Immunofluorescence, Microscopy, Quantitation Assay, Fluorescence, Centrifugation, Molecular Weight, Incubation, Enzyme-linked Immunosorbent Assay

    Differentiation and maturation of antigen presenting cells is stimulated by protein DAMPs released from irradiated tumor cells. (a) Differentiation of monocyte-derived DCs. Primary human monocytes were stimulated for 4 h with supernatants of irradiated HCC1937 cells followed by differentiation into DCs with 40 ng/ml IL-4 and 20 ng/ml GM-CSF for 5 days. Surface marker expression was measured by flow cytometry. LPS (200 ng/ml) served as positive control. x-fold increase in surface marker expression was calculated from isotype-subtracted median fluorescence intensities normalized on the corresponding 0 Gy samples. Results from 5 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (b) Biochemical characterization of the responsible molecular entities. Supernatants of 20 Gy-irradiated HCC1937 cells were subjected to membrane centrifugation (molecular weight cut-off 10 kDa) and proteinase K digestion prior to incubation with monocytes. CD80 surface expression was determined as in (a). Data from 5–10 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (c) Maturation of immature DCs. Immature DCs were differentiated from primary human monocytes with IL-4 (40 ng/ml) and GM-CSF (20 ng/ml) for 5 days. DCs were then stimulated with supernatants of irradiated HCC1937 cells for 2 days and examined by flow cytometry. TNF (100 ng/ml) served as positive control. Data from 5 independent experiments are presented, and p -values were calculated by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (d) Biochemical characterization. Prior to incubation with DCs, supernatants of 20 Gy-irradiated HCC1937 cells were applied to membrane centrifugation or proteinase K digestion, respectively, as in (b). Data from 5–10 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction.
    Figure Legend Snippet: Differentiation and maturation of antigen presenting cells is stimulated by protein DAMPs released from irradiated tumor cells. (a) Differentiation of monocyte-derived DCs. Primary human monocytes were stimulated for 4 h with supernatants of irradiated HCC1937 cells followed by differentiation into DCs with 40 ng/ml IL-4 and 20 ng/ml GM-CSF for 5 days. Surface marker expression was measured by flow cytometry. LPS (200 ng/ml) served as positive control. x-fold increase in surface marker expression was calculated from isotype-subtracted median fluorescence intensities normalized on the corresponding 0 Gy samples. Results from 5 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (b) Biochemical characterization of the responsible molecular entities. Supernatants of 20 Gy-irradiated HCC1937 cells were subjected to membrane centrifugation (molecular weight cut-off 10 kDa) and proteinase K digestion prior to incubation with monocytes. CD80 surface expression was determined as in (a). Data from 5–10 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (c) Maturation of immature DCs. Immature DCs were differentiated from primary human monocytes with IL-4 (40 ng/ml) and GM-CSF (20 ng/ml) for 5 days. DCs were then stimulated with supernatants of irradiated HCC1937 cells for 2 days and examined by flow cytometry. TNF (100 ng/ml) served as positive control. Data from 5 independent experiments are presented, and p -values were calculated by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction. (d) Biochemical characterization. Prior to incubation with DCs, supernatants of 20 Gy-irradiated HCC1937 cells were applied to membrane centrifugation or proteinase K digestion, respectively, as in (b). Data from 5–10 independent experiments are shown, and group comparison was performed by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction.

    Techniques Used: Irradiation, Derivative Assay, Marker, Expressing, Flow Cytometry, Cytometry, Positive Control, Fluorescence, Centrifugation, Molecular Weight, Incubation

    34) Product Images from "Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells"

    Article Title: Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050518

    Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR. Human epithelial cells (1×10 6 ) were pre-incubated with ( A ) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or S. cerevisiae mannan (40 µg/ml) 2 h before epithelial cells were stimulated with C. albicans walls (1×10 8 ) for 24 h. ( B ) Oral epithelial cells (3×10 5 ) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10 7 ). ( C ) Human epithelial cells (1×10 6 ) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with C. albicans walls (1×10 8 ) for 24 h ( D and E ) Cell wall mannoproteins were deproteinized by incubating C. albicans walls (1×10 8 ) with proteinase K or deglycosylated by PNGaseF digestion (cleaves N- glycosylation) or NaOH treatment (alkaline β-elimination reduces O -glycosylation). Human epithelial cells (1×10 6 ) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. ( F and G ) Epithelial cells (1×10 6 ) were incubated for 24 h with cell walls (1×10 8 ) isolated from C. albicans wild type (SC5314), N- glycosylation ( och1Δ ), O -glycosylation ( mnt1Δ/mnt2Δ ), N−/O -glycosylation ( pmr1Δ ) mutant strains or non- pathogenic S. cerevisiae . Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). ( A–G ), n = 3 (± SEM ), * p
    Figure Legend Snippet: Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR. Human epithelial cells (1×10 6 ) were pre-incubated with ( A ) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or S. cerevisiae mannan (40 µg/ml) 2 h before epithelial cells were stimulated with C. albicans walls (1×10 8 ) for 24 h. ( B ) Oral epithelial cells (3×10 5 ) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10 7 ). ( C ) Human epithelial cells (1×10 6 ) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with C. albicans walls (1×10 8 ) for 24 h ( D and E ) Cell wall mannoproteins were deproteinized by incubating C. albicans walls (1×10 8 ) with proteinase K or deglycosylated by PNGaseF digestion (cleaves N- glycosylation) or NaOH treatment (alkaline β-elimination reduces O -glycosylation). Human epithelial cells (1×10 6 ) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. ( F and G ) Epithelial cells (1×10 6 ) were incubated for 24 h with cell walls (1×10 8 ) isolated from C. albicans wild type (SC5314), N- glycosylation ( och1Δ ), O -glycosylation ( mnt1Δ/mnt2Δ ), N−/O -glycosylation ( pmr1Δ ) mutant strains or non- pathogenic S. cerevisiae . Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). ( A–G ), n = 3 (± SEM ), * p

    Techniques Used: Incubation, Isolation, Mouse Assay, Positive Control, Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

    35) Product Images from "Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes"

    Article Title: Protein features for assembly of the RNA editing helicase 2 subcomplex (REH2C) in Trypanosome holo-editosomes

    Journal: bioRxiv

    doi: 10.1101/524702

    Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30-2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30-2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30-2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T. brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA ( 27 ). Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.
    Figure Legend Snippet: Recombinant rREH2 is a catalytically active RNA helicase. (A) Purified rREH2[30-2167] samples in a denaturing gel stained with Coomassie. NTA-Ni elution fractions 1-to-3 are shown with molecular size markers (kDa). (B) Unwinding assays in 10 μL reaction mixtures with increasing concentrations of rREH2[30-2167] +/- ATP. An RNP complex (RNP) forms at the highest enzyme concentration tested; (C) Unwinding assays at increasing incubation times with rREH2[30-2167] +/- ATP using the standard reaction mixture described in the material and methods section. The standard reaction used a molar excess of enzyme over substrate. The dsRNA (ds) was assembled by pre-annealing of synthetic transcript that mimics a 3’ fragment of the T. brucei A6 pre-edited mRNA and a radiolabeled cognate guide RNA ( 27 ). Radiolabeled unwound ssRNA (ss) is indicated. Input dsRNA was heat denatured (Δ) as control. The assays in this panel were treated with proteinase K to remove any RNP that may accumulate (see methods). (D) Additional controls showing that the starting radiolabeled ssRNA used to generate the dsRNA substrate in the assay and the unwound radiolabeled ssRNA in the heat-denatured (Δ) dsRNA (in panel C) have the same gel mobility.

    Techniques Used: Recombinant, Purification, Staining, Concentration Assay, Incubation

    36) Product Images from "SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilisIdentification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae"

    Article Title: SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilisIdentification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008296

    SweD and SweC are Type II membrane proteins. ( A ) Fractionation of SweD and SweC analyzed by immunoblot. Lysates from exponentially growing wild-type cells were subject to centrifugation to separate soluble (S100) and membrane-associated (P100) proteins. The membrane fraction was incubated with buffer or buffer supplemented with TritonX-100 and the solubilized proteins (S100) were separated from insoluble material (P100) by a second round of centrifugation. Equivalent amounts of each fraction including the input protoplasts (proto) were separated by SDS-PAGE and analyzed by immunoblot. The membrane protein EzrA and cytoplasmic protein ScpB served as membrane and cytoplasmic controls. ( B ) Protease accessibility analysis of SweD and SweC analyzed by immunoblot. Protoplasts of strain BDR776 were treated with buffer or buffer supplemented with Proteinase K in the absence or presence of sodium-lauroyl-sarcosinate (Sarcosyl). Reactions were resolved by SDS-PAGE and analyzed by immunoblot. SweD and SweC were inaccessible to cleavage by Proteinase K. Degradation of the extracellular domain of SpoIVFA (FA) served as a protease accessible control. The membrane protein EzrA and the cytoplasmic protein FtsE served as protease inaccessible controls. The immunoblots shown are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.
    Figure Legend Snippet: SweD and SweC are Type II membrane proteins. ( A ) Fractionation of SweD and SweC analyzed by immunoblot. Lysates from exponentially growing wild-type cells were subject to centrifugation to separate soluble (S100) and membrane-associated (P100) proteins. The membrane fraction was incubated with buffer or buffer supplemented with TritonX-100 and the solubilized proteins (S100) were separated from insoluble material (P100) by a second round of centrifugation. Equivalent amounts of each fraction including the input protoplasts (proto) were separated by SDS-PAGE and analyzed by immunoblot. The membrane protein EzrA and cytoplasmic protein ScpB served as membrane and cytoplasmic controls. ( B ) Protease accessibility analysis of SweD and SweC analyzed by immunoblot. Protoplasts of strain BDR776 were treated with buffer or buffer supplemented with Proteinase K in the absence or presence of sodium-lauroyl-sarcosinate (Sarcosyl). Reactions were resolved by SDS-PAGE and analyzed by immunoblot. SweD and SweC were inaccessible to cleavage by Proteinase K. Degradation of the extracellular domain of SpoIVFA (FA) served as a protease accessible control. The membrane protein EzrA and the cytoplasmic protein FtsE served as protease inaccessible controls. The immunoblots shown are from one of three independent experiments. Molecular weight markers (in kDa) are indicated.

    Techniques Used: Fractionation, Centrifugation, Incubation, SDS Page, Western Blot, Molecular Weight

    37) Product Images from "Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding"

    Article Title: Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Journal: Scientific Reports

    doi: 10.1038/srep44447

    Microfluidic workflow for single cell protein profiling. ( a ) A two-inlet flow-focus droplet generator encapsulates single cells with proteinase K lysing agent into 47 μm droplets, while a one-inlet droplet maker encapsulates single barcode randomers into 53 μm droplets. (Scale Bar: 400 μm) ( b ) After thermal incubation, these droplets are controllably merged with each other and a PCR droplet using a triple-merger device. Cell and barcode droplets are introduced into two inlets, forming an interdigitated stream prior to spacing by oil (orange). One of each droplet is paired with a large PCR droplet formed upstream (green) and the three droplets electrically merged (blue). The droplets are mixed (pink) and split into two (yellow) and then four (purple) portions, followed by off-chip thermal cycling for SOE-PCR of barcodes to antibody tags.
    Figure Legend Snippet: Microfluidic workflow for single cell protein profiling. ( a ) A two-inlet flow-focus droplet generator encapsulates single cells with proteinase K lysing agent into 47 μm droplets, while a one-inlet droplet maker encapsulates single barcode randomers into 53 μm droplets. (Scale Bar: 400 μm) ( b ) After thermal incubation, these droplets are controllably merged with each other and a PCR droplet using a triple-merger device. Cell and barcode droplets are introduced into two inlets, forming an interdigitated stream prior to spacing by oil (orange). One of each droplet is paired with a large PCR droplet formed upstream (green) and the three droplets electrically merged (blue). The droplets are mixed (pink) and split into two (yellow) and then four (purple) portions, followed by off-chip thermal cycling for SOE-PCR of barcodes to antibody tags.

    Techniques Used: Incubation, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Overlap Extension Polymerase Chain Reaction

    38) Product Images from "Urb-RIP – An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins"

    Article Title: Urb-RIP – An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167877

    Urb-RIP shows Argonaute and miRNA binding to miRNA-targeted messages in human cells. a Schematic describing the let-7 reporters used to validate Urb-RIPs ability to identify interacting miRNA. b Enrichment of let-7 and mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with plasmids expressing the constructs described in a as well as a plasmid for expression of GFP-FLAG-Ago2. Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh, let-7 and GAPDH primers. c enrichment of let-7 normalized to mCh abundance in the immunoprecipitate. d western blot shows enrichment of GFP-FLAG-Ago2 following Urb-RIP. The mCh- let-7 -SLII reporters from above were co-transfected with GFP-FLAG-Ago2. Two days after transfection Urb-RIP was performed. The eluted protein as well as input was analyzed by western blot with antibody against FLAG (GFP-FLAG-Ago2 and 2HA-FLAG-Urb). Samples labeled input represent 5% of the total sample used for Urb-RIP. e quantification of western blot in d, normalized to 2HA-Urb, relative to mCh- let-7 -SLII.
    Figure Legend Snippet: Urb-RIP shows Argonaute and miRNA binding to miRNA-targeted messages in human cells. a Schematic describing the let-7 reporters used to validate Urb-RIPs ability to identify interacting miRNA. b Enrichment of let-7 and mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with plasmids expressing the constructs described in a as well as a plasmid for expression of GFP-FLAG-Ago2. Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh, let-7 and GAPDH primers. c enrichment of let-7 normalized to mCh abundance in the immunoprecipitate. d western blot shows enrichment of GFP-FLAG-Ago2 following Urb-RIP. The mCh- let-7 -SLII reporters from above were co-transfected with GFP-FLAG-Ago2. Two days after transfection Urb-RIP was performed. The eluted protein as well as input was analyzed by western blot with antibody against FLAG (GFP-FLAG-Ago2 and 2HA-FLAG-Urb). Samples labeled input represent 5% of the total sample used for Urb-RIP. e quantification of western blot in d, normalized to 2HA-Urb, relative to mCh- let-7 -SLII.

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Transfection, Expressing, Construct, Plasmid Preparation, Irradiation, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Labeling

    Urb-RIP enriches for mCherry-mRNA and bound PABP. a Schematic describing 2HA-Urb construct used for Urb-RIP and reporter, mCherry (mCh)-mRNA tagged with SLII, used to validate and optimize Urb-RIP. b Enrichment of mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with a plasmid expressing mCherry-mRNA untagged or tagged with SLII. Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh and GAPDH primers. c Comparison of relative expression amounts of mCherry with (+SLII) and without (-SLII) tagging. qPCR results show a modest reduction in mCherry expression upon insertion of SLII. Relative levels of mCherry are normalized to GAPDH. d western blot shows enrichment of PABP following Urb-RIP of mCh. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. e quantification of western blot in d . For panels b and c mean ± SD of three independent experiments are shown. For panel e mean ±SD of three independent experiments are shown. * p
    Figure Legend Snippet: Urb-RIP enriches for mCherry-mRNA and bound PABP. a Schematic describing 2HA-Urb construct used for Urb-RIP and reporter, mCherry (mCh)-mRNA tagged with SLII, used to validate and optimize Urb-RIP. b Enrichment of mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with a plasmid expressing mCherry-mRNA untagged or tagged with SLII. Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh and GAPDH primers. c Comparison of relative expression amounts of mCherry with (+SLII) and without (-SLII) tagging. qPCR results show a modest reduction in mCherry expression upon insertion of SLII. Relative levels of mCherry are normalized to GAPDH. d western blot shows enrichment of PABP following Urb-RIP of mCh. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. e quantification of western blot in d . For panels b and c mean ± SD of three independent experiments are shown. For panel e mean ±SD of three independent experiments are shown. * p

    Techniques Used: Construct, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Irradiation, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Labeling

    Urb-RIP confirms binding of PABP to BC200. a Schematic describing the BC200 construct used for pull-down by Urb-RIP. b Enrichment of BC200+SLII by Urb-RIP as determined by qPCR. A stable HEK-293 cell line for the inducible expression of 2HA-Urb was transfected with plasmids expressing the constructs described in a Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using BC200 and GAPDH primers. c Comparison of relative expression amounts of BC200 with (+SLII) and without (-SLII) tagging. qPCR results show no change in BC200 expression upon insertion of SLII. Relative levels of BC200 are normalized to GAPDH. d Western blot shows enrichment of PABP following Urb-RIP of BC200+SLII. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. e Analysis of western blot in d , normalized to 2HA-Urb, relative to mCh-SLII. For panels b and c mean ± SD of two independent experiments are shown. For panel e mean ±SD of three independent experiments are shown. * p
    Figure Legend Snippet: Urb-RIP confirms binding of PABP to BC200. a Schematic describing the BC200 construct used for pull-down by Urb-RIP. b Enrichment of BC200+SLII by Urb-RIP as determined by qPCR. A stable HEK-293 cell line for the inducible expression of 2HA-Urb was transfected with plasmids expressing the constructs described in a Two days after transfection the cells were UV-irradiated at 400 mJ/cm 2 and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using BC200 and GAPDH primers. c Comparison of relative expression amounts of BC200 with (+SLII) and without (-SLII) tagging. qPCR results show no change in BC200 expression upon insertion of SLII. Relative levels of BC200 are normalized to GAPDH. d Western blot shows enrichment of PABP following Urb-RIP of BC200+SLII. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. e Analysis of western blot in d , normalized to 2HA-Urb, relative to mCh-SLII. For panels b and c mean ± SD of two independent experiments are shown. For panel e mean ±SD of three independent experiments are shown. * p

    Techniques Used: Binding Assay, Construct, Real-time Polymerase Chain Reaction, Expressing, Transfection, Irradiation, Immunoprecipitation, Quantitative RT-PCR, Western Blot, Labeling

    39) Product Images from "The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C"

    Article Title: The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C

    Journal: BMC Biology

    doi: 10.1186/s12915-016-0332-8

    Membrane topology determination of LITAF. a Schematic diagram illustrating the LITAF protein tagged with HA at the N-terminus and myc at the C-terminus used in the experiments to determine the membrane topology. b Selective permeabilisation of the plasma membranes of HeLa cells stably expressing HA-LITAF-myc was achieved using 20 μM digitonin. Immunofluorescence microscopy was performed following incubation of the digitonin-permeabilised cells with antibodies towards HA and myc in addition to the control proteins, VPS26 and LAMP1, located on the cytosolic and luminal sides of endosomal membranes, respectively. The representative images show that both the N-terminal HA and C-terminal myc epitopes were detected concurrently with VPS26, indicating that both termini of LITAF are located on the cytosolic surface of membranes. In contrast, LAMP1 was not detected under these conditions. Permeabilsation of both plasma membrane and endosomal membranes was achieved at higher concentrations of digitonin as seen in the lower panel. c Representative graphs of a selective permeabilisation experiment illustrating the proportion of cells staining positive for HA ( top panel ) and myc ( bottom panel ) compared to VSP26 and LAMP1 (co-labelling and counting 400 cells) at increasing concentrations of digitonin. Note that all cells staining positive for HA were also positive for myc and that even at 100 μM digitonin, not all cells stained positive for LAMP1, indicating incomplete permeabilisation of endocytic vesicle membranes under these conditions. d HA-LITAF-myc was expressed using a rabbit reticulocyte lysate expression system in the presence of microsomal membranes to allow membrane insertion. A protease protection assay was then performed using proteinase K to determine the topology of the LITAF protein. In the presence of proteinase K, both N-terminal HA and C-terminal myc tags were digested, indicating that these epitopes are present on the surface of microsomal membranes ( bottom panel ). β-lactamase, containing a signal peptide that allows the translocation of a processed form to the interior of microsomal membranes, and hence protection from proteinase K digestion, was used as a control ( top panel )
    Figure Legend Snippet: Membrane topology determination of LITAF. a Schematic diagram illustrating the LITAF protein tagged with HA at the N-terminus and myc at the C-terminus used in the experiments to determine the membrane topology. b Selective permeabilisation of the plasma membranes of HeLa cells stably expressing HA-LITAF-myc was achieved using 20 μM digitonin. Immunofluorescence microscopy was performed following incubation of the digitonin-permeabilised cells with antibodies towards HA and myc in addition to the control proteins, VPS26 and LAMP1, located on the cytosolic and luminal sides of endosomal membranes, respectively. The representative images show that both the N-terminal HA and C-terminal myc epitopes were detected concurrently with VPS26, indicating that both termini of LITAF are located on the cytosolic surface of membranes. In contrast, LAMP1 was not detected under these conditions. Permeabilsation of both plasma membrane and endosomal membranes was achieved at higher concentrations of digitonin as seen in the lower panel. c Representative graphs of a selective permeabilisation experiment illustrating the proportion of cells staining positive for HA ( top panel ) and myc ( bottom panel ) compared to VSP26 and LAMP1 (co-labelling and counting 400 cells) at increasing concentrations of digitonin. Note that all cells staining positive for HA were also positive for myc and that even at 100 μM digitonin, not all cells stained positive for LAMP1, indicating incomplete permeabilisation of endocytic vesicle membranes under these conditions. d HA-LITAF-myc was expressed using a rabbit reticulocyte lysate expression system in the presence of microsomal membranes to allow membrane insertion. A protease protection assay was then performed using proteinase K to determine the topology of the LITAF protein. In the presence of proteinase K, both N-terminal HA and C-terminal myc tags were digested, indicating that these epitopes are present on the surface of microsomal membranes ( bottom panel ). β-lactamase, containing a signal peptide that allows the translocation of a processed form to the interior of microsomal membranes, and hence protection from proteinase K digestion, was used as a control ( top panel )

    Techniques Used: Stable Transfection, Expressing, Immunofluorescence, Microscopy, Incubation, Staining, Translocation Assay

    40) Product Images from "A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report"

    Article Title: A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

    Journal: BMC Neurology

    doi: 10.1186/1471-2377-10-99

    3F4 immunohistochemistry without and with proteinase K pre-treatment in the same regions of consecutive serial sections . Parallel (A, B; C, D; and E, F) cortical regions pre-treated with proteinase K (B, D, F) show marked reduction of PrP immunoreactivity when compared with serial sections without proteinase K pre-treatment (A, C, E). Different regions with variable amounts of total PrP were selected in order to have a comprehensive idea of PrP sensitivity.
    Figure Legend Snippet: 3F4 immunohistochemistry without and with proteinase K pre-treatment in the same regions of consecutive serial sections . Parallel (A, B; C, D; and E, F) cortical regions pre-treated with proteinase K (B, D, F) show marked reduction of PrP immunoreactivity when compared with serial sections without proteinase K pre-treatment (A, C, E). Different regions with variable amounts of total PrP were selected in order to have a comprehensive idea of PrP sensitivity.

    Techniques Used: Immunohistochemistry

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    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
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    Protease Inhibitor:

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana
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    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: .. After crosslinking, viruses were digested with 8U of proteinase K (NEB) at 37°C for 30 min. Crosslinked RNAs were extracted and purified with RNA Clean & Concentrator (Zymo Research) to yield RNA template for 4SU+/UV+ sequencing library sample. .. Unless otherwise mentioned, the same 4SU-containing virus without any UV irradiation was prepared in the same way to give RNA template for 4SU+/UV- control library.

    Concentration Assay:

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911
    Article Snippet: .. Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour. .. Digestion of miRNAs in ex vivo intestinal fluids; assaying miRNA levels in the small intestines in vivo Intestines from chow-fed mice were excised and flushed with 1 mL PBS to collect the contents of the intestines.

    Incubation:

    Article Title: Nucleosomes impede Cas9 access to DNA in vivo and in vitro
    Article Snippet: .. Reactions were stopped in final concentrations of 15 mM EDTA, 0.75% SDS, 150 mM NaCl, and 15 mg/mL Proteinase K and incubated at 37°C for 30 min. DNA was extracted with Phenol:Chloroform:Isoamyl Alcohol and ethanol precipitated, then resuspended in 1X CutSmart buffer and linearized by digesting with 1 U/μL DraIII-HF (New England Biolabs) at 37°C for 1 hr. ..

    Article Title: Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity
    Article Snippet: .. The permeabilised parasites were washed with DPBS, equally divided into three tubes that were incubated for 30 min on ice with either 100 μl DPBS alone (control), 100 μl DPBS containing 8 U/ml proteinase K (NEB), or 100 μl DPBS containing 0.03% saponin and 8 U/ml proteinase K, respectively. .. Reactions were quenched and proteins precipitated by adding trichloroacetic acid to 10% final concentration.

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911
    Article Snippet: .. Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour. .. Digestion of miRNAs in ex vivo intestinal fluids; assaying miRNA levels in the small intestines in vivo Intestines from chow-fed mice were excised and flushed with 1 mL PBS to collect the contents of the intestines.

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
    Article Snippet: .. Reactions were incubated at 60 °C for 15 min and stopped by adding 2.5-μL portions to 7.5 μL of 0.25 M EDTA and 1 μL of proteinase K (0.8 units, New England Biolabs) and incubating at room temperature for 30 min. After addition of 5 μL of 3x loading buffer (30% glycerol, 0.025% xylene cyanol, 0.025% bromophenol blue, 30 mM Tris, 150 mM Hepes, pH 7.5), 5 μL of the sample was loaded onto a 8% polyacrylamide gel containing 34 mM Tris, 66 mM Hepes, pH 7.5, 0.1 mM EDTA and 3 mM MgC2 The gel was then run at 4°C for 4 h at a constant power of 3 W and then dried and imaged as described above for denaturing polyacrylamide gels. .. The cDNA mimic oligonucleotide used in the native PAGE experiment ( ) consists of the R2R-G sequence fused to the reverse complement of the 50-nt acceptor lacking the terminal C residue.

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana
    Article Snippet: .. 75 µl of each sample was added to each of four tubes each containing 1 mM CaCl2 + PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 , NEB) or 1 mM CaCl2 +1% Triton X-100+ PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 ) and incubated at 25°C for 30 min. To terminate the reaction, 1 µl of protease inhibitor cocktail (Sigma-Aldrich) was added to each tube and incubated at 25°C for 10 min. 3x SDS protein loading buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 300 mM DTT, 30% glycerol, 0.3% bromophenol blue) was added and samples were boiled for 5 min before subjecting to SDS-PAGE on a 15% SDS-polyacrylamide gel. .. Immunoblot analysis with anti-GFP antibody (Invitrogen) was performed as described below.

    Sequencing:

    Article Title: Mapping RNA–capsid interactions and RNA secondary structure within virus particles using next-generation sequencing
    Article Snippet: .. After crosslinking, viruses were digested with 8U of proteinase K (NEB) at 37°C for 30 min. Crosslinked RNAs were extracted and purified with RNA Clean & Concentrator (Zymo Research) to yield RNA template for 4SU+/UV+ sequencing library sample. .. Unless otherwise mentioned, the same 4SU-containing virus without any UV irradiation was prepared in the same way to give RNA template for 4SU+/UV- control library.

    Western Blot:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: .. The resulting eluates were collected by centrifugation at 500 rcf for 2 min (at this step we always preserved 10 μl of eluate as ‘Elute’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, as described in ( )) and subsequently all proteins in the samples were digested with 0.8 U of Proteinase K (NE Biolabs) at 37°C for 30 min. .. In the case of the eIF2γ Ni2+ -pull down assay, the YMP34 strain was transformed with the selected RaP-NiP constructs along with the GCD11-His allele-carrying vector (pMP65) ( ) and the resulting transformants were cultured in the SD media and subjected to the RaP-NiP as described above.

    SDS Page:

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana
    Article Snippet: .. 75 µl of each sample was added to each of four tubes each containing 1 mM CaCl2 + PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 , NEB) or 1 mM CaCl2 +1% Triton X-100+ PK buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2 ) with or without proteinase K (200 µg ml−1 ) and incubated at 25°C for 30 min. To terminate the reaction, 1 µl of protease inhibitor cocktail (Sigma-Aldrich) was added to each tube and incubated at 25°C for 10 min. 3x SDS protein loading buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 300 mM DTT, 30% glycerol, 0.3% bromophenol blue) was added and samples were boiled for 5 min before subjecting to SDS-PAGE on a 15% SDS-polyacrylamide gel. .. Immunoblot analysis with anti-GFP antibody (Invitrogen) was performed as described below.

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    New England Biolabs proteinase k
    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.
    Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.

    Journal: Scientific Reports

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    doi: 10.1038/srep26834

    Figure Lengend Snippet: Forms of MIR2911 in cabbage extract. ( a ) Edible Plant Derived Exosome-like Nanoparticles (EPDENs) were purified from clarified cabbage lysate via ultracentrifugation. Levels of MIR2911 were quantified by qRT-PCR in various fractions: cabbage leaves liquefied in a blender (Liquefied Cabbage); Supernatant from 10,000 × g centrifugation (Slow Clarification); Supernatant from 150,000 × g ultracentrifugation (Ultra Clarification); EPDEN bands at 30% 45% interface (EPDEN band1) and 45% 60% interface (EPDEN band2) of sucrose gradient fractionation of total vesicles. ( b ) Size exclusion chromatography analysis of MIR2911 in ultracentrifugation-clarified cabbage lysate ( c ) qRT-PCR analysis of MIR2911 levels in proteinase K-treated or control (PBS-treated) ultracentrifugation-clarified cabbage lysate.

    Article Snippet: Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour.

    Techniques: Derivative Assay, Purification, Quantitative RT-PCR, Centrifugation, Clarification Assay, Fractionation, Size-exclusion Chromatography

    Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).

    Journal: Scientific Reports

    Article Title: Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    doi: 10.1038/srep26834

    Figure Lengend Snippet: Circulatory forms of MIR2911 in mice fed plant-containing diets. ( a ) Sera were obtained from mice fed either chamomile, honeysuckle, or chow diet. Exosomes were isolated from these mouse sera via ultracentrifugation protocol (UC) or PEG precipitation protocol (PEG). Levels of miR-16 and MIR2911 were quantified by qRT-PCR in the exosome pellets, or the supernatant fractions. ( b ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in control (untreated) serum samples from mice fed vegetable diets. ( c ) Time course analysis of miR-16 and MIR2911 levels in proteinase K-treated or control (untreated) serum samples from mice fed vegetable diets. ( d ) Size exclusion chromatography analysis of Let-7a, miR-16 and MIR2911 levels in proteinase K-treated serum samples from mice fed vegetable diets. Note: in Panel ( b ) and ( d ), the scale for Y axis for the 3 miRNA targets are set independently. E.g. for miR-16, the peak1 in panel ( b ) (arrow) represents similar concentration as the peak1 in panel ( d ) (arrow).

    Article Snippet: Proteinase K treatment Mouse sera or cabbage extract were mixed with proteinase K (P1807S, New England Biolabs, Ipswich, MA) at a final concentration of 1 mg/mL, or with the control PBS buffer and were incubated at 55 °C for 1 hour.

    Techniques: Mouse Assay, Isolation, Quantitative RT-PCR, Size-exclusion Chromatography, Concentration Assay

    Schematic representation of yeast  in vivo  RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+  beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the  GCN4  mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Journal: Nucleic Acids Research

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

    doi: 10.1093/nar/gkx049

    Figure Lengend Snippet: Schematic representation of yeast in vivo RNA–protein Ni 2+ -pull down (RaP-NiP) assay using formaldehyde crosslinking. The basic scheme of the RaP-NiP is described in the form of a flowchart. Green and red balls represent 40S ribosomes and eIF3 complexes, respectively, grey balls stand for the Ni 2+ beads, and purple and blue balls depict some non-specific RNA binding proteins. Exponentially growing yeast cells were crosslinked with 1% formaldehyde. Crosslinking was stopped by adding glycine and the fixed cells were lysed using glass beads by rigorous vortexing. Pre-cleared whole cell extract (WCE) containing RaP-NiP mRNAs in protein-RNA complexes were selectively digested with RNase H using sequence specific custom-made oligos. The resulting specific mRNA segments were purified with the help of the His-tagged a/TIF32 subunit of yeast eIF3 or its mutant variants using the Ni-NTA sepharose beads. Thus isolated protein-RNA complexes were subsequently treated with Proteinase K, and the captured RNAs were further purified by hot phenol extraction, reverse transcribed and their amounts were then quantified by qRT-PCR. The schematic boxed on the right-hand side illustrates typical amounts of RNAse H digested RNA segments of REI-permissive uORF1 and REI-non-permissive uORF4 from the GCN4 mRNA leader co-purifying with eIF3, the typical ratio of which is ∼4:1.

    Article Snippet: The resulting eluates were collected by centrifugation at 500 rcf for 2 min (at this step we always preserved 10 μl of eluate as ‘Elute’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, as described in ( )) and subsequently all proteins in the samples were digested with 0.8 U of Proteinase K (NE Biolabs) at 37°C for 30 min.

    Techniques: In Vivo, RNA Binding Assay, Sequencing, Purification, Mutagenesis, Isolation, Quantitative RT-PCR

    The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Erythrocyte membrane-coated nanogel for combinatorial antivirulence and responsive antimicrobial delivery against Staphylococcus aureus infection

    doi: 10.1016/j.jconrel.2017.01.016

    Figure Lengend Snippet: The formulation and characterization of RBC-nanogels. (A) RBC-nanogels loaded with rhodamine B were formulated and subjected to (i) no treatment, (ii) treated with Triton X-100 and proteinase K, or (iii) Triton X-100 and proteinase K followed by tris(2-carboxyethyl) phosphine (TCEP). The RBC-nanogels were then filtered to collect the released dye, which was further measured by a UV-vis spectrophotometer. (B) A representative TEM image of RBC-nanogels (scale bar = 100 nm). (C) Dynamic light scattering (DLS) measurements of the size and size distribution of RBC-vesicles, RBC-nanogels, and non-responsive RBC nanogels (Control nanogels) subjected to the same treatment as in (A).

    Article Snippet: Following the nanogel synthesis, samples were added with 0.25 mL 5% w/v Triton X-100 and 20 µg proteinase K (New England Biolabs, Inc., Beverly, USA) to dissolve RBC membranes.

    Techniques: Spectrophotometry, Transmission Electron Microscopy

    Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

    Journal: bioRxiv

    Article Title: TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

    doi: 10.1101/299727

    Figure Lengend Snippet: Schematic overview of TAF-ChIP approach. (1) Formaldehyde fixed cells were directly sorted into RIPA buffer (see methods for details). (2) Cells were briefly sonicated at low intensity to break open the nuclei. (3) Antibodies were coupled to magnetic beads in the presence of blocking reagents. (4) Antibody coupled beads were added to the cell lysate and incubated overnight at 4°C. (5) The tagmentation reaction was performed after initial washes with low salt IP buffer and homemade tagmentation buffer. (6) The tagmentation reaction and the background regions (not anchored by antibody interaction) were washed away with subsequent high stringency washes. (7) The proteinase K was heat-inactivated and the material was PCR-amplified without purification.

    Article Snippet: The samples were subjected to proteinase K treatment in a 50 μl of TE buffer pH 8.0 with 5 μl of 20 mg/ml of proteinase K. The proteinase K was heat inactivated for 95 °C for 5 min in a 100 μl reaction with 1X NEBNext High-Fidelity PCR Mix, during PCR with primers containing molecular indices (listed in Table 1) with the following program; 72°C for 3min, 95°C for 5min, {98°C for 10 sec, 63°C for 30 sec, 72°C for 30 sec} for 12 cycles, 72°C for 5 min, and hold at 4°C.

    Techniques: Chromatin Immunoprecipitation, Sonication, Magnetic Beads, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Purification