proteinase k  (TaKaRa)


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    Name:
    Proteinase K
    Description:
    Proteinase K RNase A rDNase Set and Protein Quantification Assay
    Catalog Number:
    740506
    Price:
    None
    Category:
    Enzymes and protein quantification Buffers enzymes Accessories and components Nucleic acid purification
    Size:
    100 mg
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    Structured Review

    TaKaRa proteinase k
    RNase protection assay of siRNA in lipoplexes and LNPs diluted with HBSS. Notes:  After addition of RNase to samples, mixtures were incubated at 37°C for 1 h (siRNA: 1.25 µM). The reaction was terminated by the addition of 5 µL proteinase K. Almost 100% of the siRNA remained in LNPs, whereas 80% of the siRNA remained in lipoplexes. The student’s  t -test was performed. * P
    Proteinase K RNase A rDNase Set and Protein Quantification Assay
    https://www.bioz.com/result/proteinase k/product/TaKaRa
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation"

    Article Title: Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S136426

    RNase protection assay of siRNA in lipoplexes and LNPs diluted with HBSS. Notes:  After addition of RNase to samples, mixtures were incubated at 37°C for 1 h (siRNA: 1.25 µM). The reaction was terminated by the addition of 5 µL proteinase K. Almost 100% of the siRNA remained in LNPs, whereas 80% of the siRNA remained in lipoplexes. The student’s  t -test was performed. * P
    Figure Legend Snippet: RNase protection assay of siRNA in lipoplexes and LNPs diluted with HBSS. Notes: After addition of RNase to samples, mixtures were incubated at 37°C for 1 h (siRNA: 1.25 µM). The reaction was terminated by the addition of 5 µL proteinase K. Almost 100% of the siRNA remained in LNPs, whereas 80% of the siRNA remained in lipoplexes. The student’s t -test was performed. * P

    Techniques Used: Rnase Protection Assay, Incubation

    2) Product Images from "Leptospiral Hemolysins Induce Proinflammatory Cytokines through Toll-Like Receptor 2-and 4-Mediated JNK and NF-?B Signaling Pathways"

    Article Title: Leptospiral Hemolysins Induce Proinflammatory Cytokines through Toll-Like Receptor 2-and 4-Mediated JNK and NF-?B Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042266

    Expression, purification and hemolytic activity of rL-hemolysin proteins. (A). Hemolysin genes amplified from genomic DNA of L. interrogans strain Lai. Lane AM: DNA marker (Fermentas, Canada). Lane A1: blank control. Lanes A2 to A9: amplicons of the sph1 (1674 bp), sph2 (1869 bp), sph3 (1557 bp), sph4 (717 bp), hlpA (939 bp), hlyC (1332 bp), hlyX (1176 bp) and tlyA (828 bp) genes from L. interrogans strain Lai. (B). Expression of the rL-hemolysin proteins. Lane BM: protein marker (Sangon Biotech, China). Lane B1: wild-type pET42a. Lanes B2 to B9: expressed recombinant proteins of rSph1 (63.5 kDa), rSph2 (71.1 kDa), rSph3 (60.7 kDa), rSph4 (27.9 kDa), rHlpA (36.5 kDa), rHlyC (50.4 kDa), rHlyX (43.1 kDa) and rTlyA (31.5 kDa). (C). Purification of the rL-hemolysin proteins. Lane CM: protein marker (Sangon Biotech). Lane C1: blank control. Lanes C2 to C9: purified rSph1, rSph2, rSph3, rSph4, rHlpA, rHlyC, rHlyX and rTlyA proteins. (D). Hemolytic activity of the rL-hemolysin proteins measured by spectrophotometry. Bars show the mean ± SD of three independent experiments. PK-H indicates that the rL-hemolysins were pretreated with proteinase K (PK) digestion plus heat-inactivation. The A420 values from spectrophotometric measurement at 420 nm reflect the levels of hemoglobin released from sheep erythrocytes. The A420 value of the supernatant from the sheep erythrocytes in 1 mL 5% erythrocyte suspension lysed by distilled water was set at 100% (total hemolysis). rOmpL1, a non-hemolytic recombinant porin from L. interrogans strain Lai, was the negative control (background hemolysis). Relative hemolytic activity of each of the rL-hemolysin proteins was defined as the percentage (%, A420) compared to total hemolysis. * P
    Figure Legend Snippet: Expression, purification and hemolytic activity of rL-hemolysin proteins. (A). Hemolysin genes amplified from genomic DNA of L. interrogans strain Lai. Lane AM: DNA marker (Fermentas, Canada). Lane A1: blank control. Lanes A2 to A9: amplicons of the sph1 (1674 bp), sph2 (1869 bp), sph3 (1557 bp), sph4 (717 bp), hlpA (939 bp), hlyC (1332 bp), hlyX (1176 bp) and tlyA (828 bp) genes from L. interrogans strain Lai. (B). Expression of the rL-hemolysin proteins. Lane BM: protein marker (Sangon Biotech, China). Lane B1: wild-type pET42a. Lanes B2 to B9: expressed recombinant proteins of rSph1 (63.5 kDa), rSph2 (71.1 kDa), rSph3 (60.7 kDa), rSph4 (27.9 kDa), rHlpA (36.5 kDa), rHlyC (50.4 kDa), rHlyX (43.1 kDa) and rTlyA (31.5 kDa). (C). Purification of the rL-hemolysin proteins. Lane CM: protein marker (Sangon Biotech). Lane C1: blank control. Lanes C2 to C9: purified rSph1, rSph2, rSph3, rSph4, rHlpA, rHlyC, rHlyX and rTlyA proteins. (D). Hemolytic activity of the rL-hemolysin proteins measured by spectrophotometry. Bars show the mean ± SD of three independent experiments. PK-H indicates that the rL-hemolysins were pretreated with proteinase K (PK) digestion plus heat-inactivation. The A420 values from spectrophotometric measurement at 420 nm reflect the levels of hemoglobin released from sheep erythrocytes. The A420 value of the supernatant from the sheep erythrocytes in 1 mL 5% erythrocyte suspension lysed by distilled water was set at 100% (total hemolysis). rOmpL1, a non-hemolytic recombinant porin from L. interrogans strain Lai, was the negative control (background hemolysis). Relative hemolytic activity of each of the rL-hemolysin proteins was defined as the percentage (%, A420) compared to total hemolysis. * P

    Techniques Used: Expressing, Purification, Activity Assay, Amplification, Marker, Recombinant, Spectrophotometry, Negative Control

    Blocking effects of TLR-IgGs and TLR2 or TLR4 deficieincy on the production of IL-1β, IL-6 and TNF-α under induction of the rL-hemolysins for 24 h. Bars show the mean± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat–inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and were used to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. The concentration of each of the rL-hemolysins or E-LPS tesed was 1 µg. The mouse monocytes were separated from the peripheral blood samples from TLR2 −/− , TLR4 −/− or TLR2,4 −/− C57BL/6 mice. Control indicates the IL-1β, IL-6 and TNF-α levels in the human THP-1, mouse J774A.1 macrophages, and primary mouse monocytes from wild-type C57BL/6 without TLR2-, TLR4-or TLR2,4-deficience before treatement with any rL-hemolysins or E-LPS. # P
    Figure Legend Snippet: Blocking effects of TLR-IgGs and TLR2 or TLR4 deficieincy on the production of IL-1β, IL-6 and TNF-α under induction of the rL-hemolysins for 24 h. Bars show the mean± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat–inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and were used to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. The concentration of each of the rL-hemolysins or E-LPS tesed was 1 µg. The mouse monocytes were separated from the peripheral blood samples from TLR2 −/− , TLR4 −/− or TLR2,4 −/− C57BL/6 mice. Control indicates the IL-1β, IL-6 and TNF-α levels in the human THP-1, mouse J774A.1 macrophages, and primary mouse monocytes from wild-type C57BL/6 without TLR2-, TLR4-or TLR2,4-deficience before treatement with any rL-hemolysins or E-LPS. # P

    Techniques Used: Blocking Assay, Concentration Assay, Mouse Assay

    Ability of the rL-hemolysin proteins to induce IL-1β, IL-6 and TNF-α in human and mouse macrophages. (A). IL-1β, IL-6 and TNF-α levels in human THP-1 and mouse J774A.1 macrophages induced by each of the rL-hemolysin proteins for 24 h with the indicated protein concentrations. Bars show the mean ± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat-inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and used as controls to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. Controls indicate the IL-1β, IL-6 and TNF-α levels in the THP-1 or J774A.1 macrophages before treatment with any rL-hemolysins or E-LPS. E-LPS indicates the LPS of E. coli serotype O111:B4. * P
    Figure Legend Snippet: Ability of the rL-hemolysin proteins to induce IL-1β, IL-6 and TNF-α in human and mouse macrophages. (A). IL-1β, IL-6 and TNF-α levels in human THP-1 and mouse J774A.1 macrophages induced by each of the rL-hemolysin proteins for 24 h with the indicated protein concentrations. Bars show the mean ± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat-inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and used as controls to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. Controls indicate the IL-1β, IL-6 and TNF-α levels in the THP-1 or J774A.1 macrophages before treatment with any rL-hemolysins or E-LPS. E-LPS indicates the LPS of E. coli serotype O111:B4. * P

    Techniques Used:

    JNK and NF-κB pathways mediate the production of IL-1β, IL-6 and TNF-α under induction of the rL-hemolysins for 24 h. Bars show the mean ± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat-inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and were used to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. The concentration of each of the rL-hemolysins or E-LPS tesed was 1 µg. Control indicates the IL-1β, IL-6 and TNF-α levels in the human THP-1 and mouse J774A.1 macrophages before treatment with any rL-hemolysins or E-LPS. # P
    Figure Legend Snippet: JNK and NF-κB pathways mediate the production of IL-1β, IL-6 and TNF-α under induction of the rL-hemolysins for 24 h. Bars show the mean ± SD of three independent experiments. E-LPS indicates the LPS of E. coli serotype O111:B4. PK-H indicates that the rL-hemolysins or E-LPS were pretreated with proteinase K digestion plus heat-inactivation while PMB indicates that the rL-hemolysins or E-LPS were pretreated with polymyxin B blockade, and were used to monitor possible contamination with E. coli LPS in the rL-hemolysin proteins. The concentration of each of the rL-hemolysins or E-LPS tesed was 1 µg. Control indicates the IL-1β, IL-6 and TNF-α levels in the human THP-1 and mouse J774A.1 macrophages before treatment with any rL-hemolysins or E-LPS. # P

    Techniques Used: Concentration Assay

    Related Articles

    Incubation:

    Article Title: Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors
    Article Snippet: .. The cultures were then diluted 1:200 with TSBG supplemented with or without nicotine, and 200 μL of bacterial suspension was added to each well of a 96-well microplate and incubated at 37 °C for 24 h. To determine the effect of DNase I and proteinase K on biofilm formation, 5 μL DNase I (5 U/μL, Takara, Shanghai, China) and 2 μg/mL Proteinase K (Sango, Shanghai, China) were added to the wells. .. Thereafter, the wells were washed three times with phosphate-buffered saline (PBS) to remove unattached bacteria and then 200 μL of 100% methanol was added to each well to fix the attached cells at room temperature for 20 min. After removal of the methanol, the biofilms were air-dried and stained with 2% crystal violet at room temperature for 8 min.

    Polymerase Chain Reaction:

    Article Title: Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum
    Article Snippet: L. plantarum ST-III (CGMCC No. 0847) used in this study was cultured in MRS broth at 37 °C (Merck KGaA, Darmstadt, Germany). .. The starting ssDNA library and the PCR primers used for PCR amplifications were supplied by Integrated DNA Technologies (IDT, Coralville, IA, USA); tRNA, DMSO and streptavidin-HRP (horseradish peroxidase) were purchased from Sigma (St. Louis, MO, USA); BSA and all PCR chemicals were ordered from Invitrogen China (Shanghai, China); trypsin and proteinase K were purchased from TaKaRa (TaKaRa, Dalian, China); and DNA-BIND 96-well plates were obtained from Corning (Corning, New York, NY, USA). .. Water was filtered with a Milli-Q water purification system (Millipore, Bedford, MA, USA).

    Titration:

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1
    Article Snippet: .. The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals. .. The starting concentration in these experiments was 200 μM [15 N]RPA32172–270 or 250 μM [15 N]SMARCAL11–32 .

    Labeling:

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1
    Article Snippet: .. The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals. .. The starting concentration in these experiments was 200 μM [15 N]RPA32172–270 or 250 μM [15 N]SMARCAL11–32 .

    Binding Assay:

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1
    Article Snippet: .. The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals. .. The starting concentration in these experiments was 200 μM [15 N]RPA32172–270 or 250 μM [15 N]SMARCAL11–32 .

    In Situ:

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1
    Article Snippet: .. The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals. .. The starting concentration in these experiments was 200 μM [15 N]RPA32172–270 or 250 μM [15 N]SMARCAL11–32 .

    Nuclear Magnetic Resonance:

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1
    Article Snippet: .. The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals. .. The starting concentration in these experiments was 200 μM [15 N]RPA32172–270 or 250 μM [15 N]SMARCAL11–32 .

    Concentration Assay:

    Article Title: TDP-43 stabilises the processing intermediates of mitochondrial transcripts
    Article Snippet: For Proteinase K protection assay, the isolated mitochonaria was diluted into 1 mg/ml with Solution B (10 mM Hepes-KOH pH7.6, 500 mM Sucrose). .. 100 µg of mitochondria was treated with the indicated concentration of Proteinase K (Takara Bio, Osaka, Japan) for 20 min on ice. ..

    Footprinting:

    Article Title: Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control
    Article Snippet: .. For dimethyl sulfate (DMS) footprinting, native gel electrophoresis and photocleavage footprinting, the samples were further treated with 0.4 mg/ml of RNase A (Fermentas, MBI), then 0.6 mg/ml proteinase K (TAKARA, Dalian) at 37°C for 1 h each. .. Analysis of RNA transcripts Transcription was performed as described earlier in the text, except that 20 ng/µl of plasmid DNA was used as the transcription template, 1 U/µl of RiboLock RNase inhibitor (Fermentas, MBI) was added and 10% of the UTP was substituted with fluorescein-UTP (Fermentas, MBI).

    Nucleic Acid Electrophoresis:

    Article Title: Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control
    Article Snippet: .. For dimethyl sulfate (DMS) footprinting, native gel electrophoresis and photocleavage footprinting, the samples were further treated with 0.4 mg/ml of RNase A (Fermentas, MBI), then 0.6 mg/ml proteinase K (TAKARA, Dalian) at 37°C for 1 h each. .. Analysis of RNA transcripts Transcription was performed as described earlier in the text, except that 20 ng/µl of plasmid DNA was used as the transcription template, 1 U/µl of RiboLock RNase inhibitor (Fermentas, MBI) was added and 10% of the UTP was substituted with fluorescein-UTP (Fermentas, MBI).

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    TaKaRa proteinase k
    HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K +  and 40% (w/v) PEG 200.
    Proteinase K, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/TaKaRa
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    HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K +  and 40% (w/v) PEG 200.

    Journal: Nucleic Acids Research

    Article Title: Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control

    doi: 10.1093/nar/gkt264

    Figure Lengend Snippet: HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K + and 40% (w/v) PEG 200.

    Article Snippet: For dimethyl sulfate (DMS) footprinting, native gel electrophoresis and photocleavage footprinting, the samples were further treated with 0.4 mg/ml of RNase A (Fermentas, MBI), then 0.6 mg/ml proteinase K (TAKARA, Dalian) at 37°C for 1 h each.

    Techniques: Footprinting, Labeling, Nucleic Acid Electrophoresis, Sequencing, Mutagenesis

    Binding assays of aptamer CCFM641-5 to trypsin-treated or proteinase K-treated  B. bifidum . ( A ) Flow cytometric analysis of aptamer CCFM641-5 binding affinity for trypsin-treated  B. bifidum ; ( B ) Flow cytometric analysis of aptamer CCFM641-5 binding ability to proteinase K-treated  B. bifidum .

    Journal: International Journal of Molecular Sciences

    Article Title: Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum

    doi: 10.3390/ijms18050883

    Figure Lengend Snippet: Binding assays of aptamer CCFM641-5 to trypsin-treated or proteinase K-treated B. bifidum . ( A ) Flow cytometric analysis of aptamer CCFM641-5 binding affinity for trypsin-treated B. bifidum ; ( B ) Flow cytometric analysis of aptamer CCFM641-5 binding ability to proteinase K-treated B. bifidum .

    Article Snippet: The starting ssDNA library and the PCR primers used for PCR amplifications were supplied by Integrated DNA Technologies (IDT, Coralville, IA, USA); tRNA, DMSO and streptavidin-HRP (horseradish peroxidase) were purchased from Sigma (St. Louis, MO, USA); BSA and all PCR chemicals were ordered from Invitrogen China (Shanghai, China); trypsin and proteinase K were purchased from TaKaRa (TaKaRa, Dalian, China); and DNA-BIND 96-well plates were obtained from Corning (Corning, New York, NY, USA).

    Techniques: Binding Assay, Flow Cytometry

    Topology of the UL56 protein. HSV-2-infected cells (a), virions (b), and UL56-expressing cells (c) were treated with proteinase K in either the presence (+) or the absence (−) of 1% Triton X-100. The samples were then separated by SDS-PAGE and analyzed by Western blotting with anti-UL56 serum. Positions of molecular mass markers (in kilodaltons) are indicated on the left.

    Journal: Journal of Virology

    Article Title: Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2

    doi: 10.1128/JVI.76.13.6718-6728.2002

    Figure Lengend Snippet: Topology of the UL56 protein. HSV-2-infected cells (a), virions (b), and UL56-expressing cells (c) were treated with proteinase K in either the presence (+) or the absence (−) of 1% Triton X-100. The samples were then separated by SDS-PAGE and analyzed by Western blotting with anti-UL56 serum. Positions of molecular mass markers (in kilodaltons) are indicated on the left.

    Article Snippet: After the cells were homogenized by 10 strokes with a glass homogenizer, proteinase K (TaKaRa) was added to cell homogenates in the presence or absence of 1% Triton X-100.

    Techniques: Infection, Expressing, SDS Page, Western Blot

    (A)  15 N– 1 H HMQC spectra of RPA32C.  15 N– 1 H SOFAST HMQC spectra of RPA32 172–270  before (black) and after digestion with (red) Proteinase K. The most N-terminal residue not perturbed by the protease, A202, is highlighted by the circle. (B) Superposition of the  15 N– 1 H SOFAST HMQC spectra of RPA32 172–270  (black) and RPA32C (red). (C) A select region of the spectrum with multiple time points to demonstrate residues that are either protected (F248) or digested (F199).

    Journal: Biochemistry

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1

    doi: 10.1021/bi500252w

    Figure Lengend Snippet: (A) 15 N– 1 H HMQC spectra of RPA32C. 15 N– 1 H SOFAST HMQC spectra of RPA32 172–270 before (black) and after digestion with (red) Proteinase K. The most N-terminal residue not perturbed by the protease, A202, is highlighted by the circle. (B) Superposition of the 15 N– 1 H SOFAST HMQC spectra of RPA32 172–270 (black) and RPA32C (red). (C) A select region of the spectrum with multiple time points to demonstrate residues that are either protected (F248) or digested (F199).

    Article Snippet: The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals.

    Techniques:

    NMR analysis of [ 15 N]SMARCAL1 with RPA32C. (A) 15 N– 1 H SOFAST HMQC spectra of [ 15 N]SMARCAL1 1–32 in the absence (black) and presence (red) of RPA32C. (B) 15 N– 1 H SOFAST HMQC spectra of [ 15 N]SMARCAL1 1–32 in complex with RPA32C obtained before (black) and after (red) a 50 min Proteinase K digestion.

    Journal: Biochemistry

    Article Title: Structural Analysis of Replication Protein A Recruitment of the DNA Damage Response Protein SMARCAL1

    doi: 10.1021/bi500252w

    Figure Lengend Snippet: NMR analysis of [ 15 N]SMARCAL1 with RPA32C. (A) 15 N– 1 H SOFAST HMQC spectra of [ 15 N]SMARCAL1 1–32 in the absence (black) and presence (red) of RPA32C. (B) 15 N– 1 H SOFAST HMQC spectra of [ 15 N]SMARCAL1 1–32 in complex with RPA32C obtained before (black) and after (red) a 50 min Proteinase K digestion.

    Article Snippet: The chemical shift perturbations in a titration of labeled RPA32C with SMARCAL17–32 were analyzed using a weighted average of the change in chemical shift (Δδ) upon binding based on the net perturbations in both the 1 H and 15 N dimensions calculated using the standard equation (eq ): 1 For in situ proteolysis experiments, 1 μL of Proteinase K (Clontech) dissolved at a ratio of 1/100 (w/v) was added to NMR samples and SOFAST-HMQC spectra were collected at 10 min intervals.

    Techniques: Nuclear Magnetic Resonance