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
    Price:
    81
    Size:
    2 ml
    Category:
    Proteases
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    Structured Review

    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 90 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) Product Images from "5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells"

    Article Title: 5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky444

    Primer extension assays for replication bypass of reductively stabilized DNA–protein or DNA–peptide cross-links by hPol η (top panel) and hPol κ (bottom panel). 32P-labeled 12-mer primer was annealed with 23-mer template containing unmodified dC ( A ), 5-formyl-dC ( B ), or C5-dC cross-links to 11-mer peptide RPKPQQFFGLM-CONH2 ( C ) 31-mer peptide YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE ( D ), full size histone H2A ( E ), histone H4 ( F ), or proteinase K digested histone H4 ( G ). Polymerase reactions were initiated by addition of DNA polymerases and a mixture of dNTP and quenched at pre-selected time points prior to loading onto 20% denaturing PAGE.
    Figure Legend Snippet: Primer extension assays for replication bypass of reductively stabilized DNA–protein or DNA–peptide cross-links by hPol η (top panel) and hPol κ (bottom panel). 32P-labeled 12-mer primer was annealed with 23-mer template containing unmodified dC ( A ), 5-formyl-dC ( B ), or C5-dC cross-links to 11-mer peptide RPKPQQFFGLM-CONH2 ( C ) 31-mer peptide YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE ( D ), full size histone H2A ( E ), histone H4 ( F ), or proteinase K digested histone H4 ( G ). Polymerase reactions were initiated by addition of DNA polymerases and a mixture of dNTP and quenched at pre-selected time points prior to loading onto 20% denaturing PAGE.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis

    6) Product Images from "5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells"

    Article Title: 5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky444

    Primer extension assays for replication bypass of reductively stabilized DNA–protein or DNA–peptide cross-links by hPol η (top panel) and hPol κ (bottom panel). 32P-labeled 12-mer primer was annealed with 23-mer template containing unmodified dC ( A ), 5-formyl-dC ( B ), or C5-dC cross-links to 11-mer peptide RPKPQQFFGLM-CONH2 ( C ) 31-mer peptide YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE ( D ), full size histone H2A ( E ), histone H4 ( F ), or proteinase K digested histone H4 ( G ). Polymerase reactions were initiated by addition of DNA polymerases and a mixture of dNTP and quenched at pre-selected time points prior to loading onto 20% denaturing PAGE.
    Figure Legend Snippet: Primer extension assays for replication bypass of reductively stabilized DNA–protein or DNA–peptide cross-links by hPol η (top panel) and hPol κ (bottom panel). 32P-labeled 12-mer primer was annealed with 23-mer template containing unmodified dC ( A ), 5-formyl-dC ( B ), or C5-dC cross-links to 11-mer peptide RPKPQQFFGLM-CONH2 ( C ) 31-mer peptide YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE ( D ), full size histone H2A ( E ), histone H4 ( F ), or proteinase K digested histone H4 ( G ). Polymerase reactions were initiated by addition of DNA polymerases and a mixture of dNTP and quenched at pre-selected time points prior to loading onto 20% denaturing PAGE.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis

    7) 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

    8) 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

    9) Product Images from "Reprogramming an ATP-driven protein machine into a light-gated nanocage"

    Article Title: Reprogramming an ATP-driven protein machine into a light-gated nanocage

    Journal: Nature nanotechnology

    doi: 10.1038/nnano.2013.242

    xMm-cpn is a protease-resistant nanocage and can be used for light-triggered release of non-native cargos. a Unlit closed xMm-cpn is protected against proteinase K (PK, scissors symbol). A SDS-PAGE gel of unlit and blue light-illuminated xMm-cpn after PK incubation shows the appearance of smaller molecular weight fragments only after light-induced cage opening. b Blue light-illumination renders the C-terminal 6His-tag (half circle) of xMm-cpn accessible for binding of Ni-NTA coupled probes (yellow sphere): Unlit and blue light illuminated xMm-cpn incubated with either (i) Ni-NTA-nanogold (Au) or (ii) fluorescent Ni-NTA-Atto647N (dye) analysed on non-denaturing PAGE gels. The protein is visualized by coomassie staining (left panels) while the Ni-NTA-compounds are visualized (right panels) using either gold specific stain (Ni-NTA-nanogold) or via their fluorescence (Ni-NTA-Atto647N). Note the co-localization of Ni-NTA-Atto647N with both open and closed states, whereas Ni-NTA-nanogold is only co-localized to the open state. c Ni-NTA-Atto647N (red star) bound to the C-terminal 6His-tag of xMm-cpn in open and closed states stays bound to xMm-cpn in the presence of PK in the closed state, but is cleaved off from the cage after blue light-illumination (disappearance of the cargo band in rightmost lane) as analysed on a non-denaturing PAGE gel. The cargo (Ni-NTA-Atto647N) is visualized by its fluorescence (bottom gel panel).
    Figure Legend Snippet: xMm-cpn is a protease-resistant nanocage and can be used for light-triggered release of non-native cargos. a Unlit closed xMm-cpn is protected against proteinase K (PK, scissors symbol). A SDS-PAGE gel of unlit and blue light-illuminated xMm-cpn after PK incubation shows the appearance of smaller molecular weight fragments only after light-induced cage opening. b Blue light-illumination renders the C-terminal 6His-tag (half circle) of xMm-cpn accessible for binding of Ni-NTA coupled probes (yellow sphere): Unlit and blue light illuminated xMm-cpn incubated with either (i) Ni-NTA-nanogold (Au) or (ii) fluorescent Ni-NTA-Atto647N (dye) analysed on non-denaturing PAGE gels. The protein is visualized by coomassie staining (left panels) while the Ni-NTA-compounds are visualized (right panels) using either gold specific stain (Ni-NTA-nanogold) or via their fluorescence (Ni-NTA-Atto647N). Note the co-localization of Ni-NTA-Atto647N with both open and closed states, whereas Ni-NTA-nanogold is only co-localized to the open state. c Ni-NTA-Atto647N (red star) bound to the C-terminal 6His-tag of xMm-cpn in open and closed states stays bound to xMm-cpn in the presence of PK in the closed state, but is cleaved off from the cage after blue light-illumination (disappearance of the cargo band in rightmost lane) as analysed on a non-denaturing PAGE gel. The cargo (Ni-NTA-Atto647N) is visualized by its fluorescence (bottom gel panel).

    Techniques Used: SDS Page, Incubation, Molecular Weight, Binding Assay, Polyacrylamide Gel Electrophoresis, Staining, Fluorescence

    10) 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

    11) Product Images from "Lipids and Proteins Act in Opposing Manners To Regulate Polyomavirus Infection ▿"

    Article Title: Lipids and Proteins Act in Opposing Manners To Regulate Polyomavirus Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01093-10

    Removal of plasma membrane glycoproteins stimulates Py infection and ER transport. (A) NIH 3T3 cells were treated with 4 mg of proteinase K/ml at 4°C for 1 h or left untreated. The contents of media from these cells were precipitated and subjected
    Figure Legend Snippet: Removal of plasma membrane glycoproteins stimulates Py infection and ER transport. (A) NIH 3T3 cells were treated with 4 mg of proteinase K/ml at 4°C for 1 h or left untreated. The contents of media from these cells were precipitated and subjected

    Techniques Used: Infection

    12) 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

    13) 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

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    Amplification:

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    Lambda DNA Preparation:

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    Incubation:

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    Gel Extraction:

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    Expressing:

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    Modification:

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    Western Blot:

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    Pulse Chase:

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    Concentration Assay:

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    Methylation:

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    Generated:

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    other:

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    Imaging:

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    Polymerase Chain Reaction:

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    Article Snippet: A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo). .. Libraries were PCR amplified with Q5 master mix (NEB) for 6–18 cycles (chosen by performing qPCR on the pre-amplified library).

    Article Title: Base-resolution profiling of active DNA demethylation using methylase-assisted bisulfite sequencing (MAB-seq) and caMAB-seq
    Article Snippet: Sodium acetate buffer solution (3 M, pH 5.2±0.1; Sigma-Aldrich, cat. no. S7899) EDTA (0.5 M, pH8.0; Life Technologies, cat. no. 15575-020) EGTA (0.5 M, pH8.0; prepared from Sigma-Aldrich, cat. no. 03777) RNase A (10 mg/mL; Life Technologies, cat. no. EN0531) Anti-H3K4me1 (10 μg/mL; Abcam, cat. no. ab8895) TaqαI (20 U/μL; New England Biolabs, cat. no. R0149L) Klenow fragment (exo– , 5 U/μL; Thermo Scientific, cat. no. EP0422) T4 DNA ligase (2000 U/μL; New England Biolabs, cat. no. M0202M) CutSmart buffer (10×; New England Biolabs, cat. no. B7204S) ATP (100 mM; Thermo Scientific, cat. no. R0441) dNTP set 100 mM solutions (Life Technologies, cat. no. R0181) SPRIselect reagent kit (Beckman Coulter, cat. no. ) DNeasy blood & tissue kit (Qiagen, cat. no. 69504) Qiagen EpiTect DNA bisulfite kit (Qiagen, cat. no. 59104) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) MinElute PCR purification kit (Qiagen, cat. no. 28004) M.SssI (20 U/μL, New England Biolabs, M0226M) NEBuffer 2 (10x; cat. no. B7002S) S-adenosylmethionine (SAM) (32 mM; New England Biolabs, cat. no. B9003S) CRITICAL Always use SAM before its expiration date and make aliquots to avoid multiple cycles of freeze / thaw. .. Unmethylated Lambda DNA (Promega, cat. no. D1521) Agilent High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067-4626) cOmplete EDTA-free proteinase inhibitor (Roche, cat. no. 11873580001) Dynabeads Protein G for Immunoprecipitation (ThermoFisher Scientific, cat. no. 10004D) RNase A (Life Technologies, cat. no. 12091-201) Proteinase K (New England Biolabs, cat. no. P8107S) Glycogen (Roche Life Science, cat. no. 10901393001) 5 PRIME Phase Lock Gel heavy 2 mL (Fisher Scientific, cat. no. FP2302830) Phenol - chloroform - isoamyl alcohol mixture (Sigma-Aldrich, cat. no. 77617) !

    Article Title: Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm
    Article Snippet: Genomic DNA from a right ear clip was used for PCR genotyping. .. Ear clips were lysed overnight at 60°C in a solution with tail lysis buffer (100 mM Tris-HCl pH 8.1, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) supplemented with Proteinase K (NEB, Cat. # P8107S).

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: .. After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions. .. In all, 1/30 of the purified DNA was analyzed by qPCR using the Power SYBR™ Green PCR Master Mix (cat.#4367659, Life Technologies).

    Sonication:

    Article Title: Post-conversion sialylation of prions in lymphoid tissues
    Article Snippet: We diluted 10% (wt/vol) 263K brain homogenates 10-fold in 25 mM Tris, pH 7.4, buffer supplied with 1% (wt/vol) Triton X-100 and sonicated for 30 s at 170 W energy output in the microplate horn (Misonix S-4000). .. The samples were then incubated with PK (10 µg/mL, 37 °C, 1 h) (cat. no. P8107S; New England Biolabs).

    In Vivo:

    Article Title: Nucleotide excision repair by dual incisions in plants
    Article Snippet: Paragraph title: In Vivo Excision Repair Assay. ... Arabidopsis cell lysates were incubated with RNase A (1:500; R4642 Sigma) for 10 min at room temperature and then deproteinized by the addition of SDS to a final concentration of 0.33% (from a 10% stock) and incubation with Proteinase K (1:400; P8107S New England Biolabs) for 30 min at 55 °C.

    Ion Exchange Chromatography:

    Article Title: Post-conversion sialylation of prions in lymphoid tissues
    Article Snippet: Paragraph title: Ion-Exchange Chromatography. ... The samples were then incubated with PK (10 µg/mL, 37 °C, 1 h) (cat. no. P8107S; New England Biolabs).

    Magnetic Beads:

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: After O/N incubation of the chromatin with the antibodies at 4 °C, the immunocomplexes were captured with 50 μl of Protein A magnetic beads (cat.#10001D, Invitrogen) (beads were pre-blocked with 5% IgG-free bovine serum albumin (cat.#001-000-161, Jackson ImmunoResearch Laboratories)) for further 4 h at 4 °C. .. After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions.

    Isolation:

    Article Title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins
    Article Snippet: .. A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo). .. RNA was reverse transcribed with AffinityScript (Agilent), treated with ExoSap-IT (Affymetrix) to remove excess oligonucleotides, and a DNA adapter was ligated to the 3′ end using T4 RNA Ligase I (NEB) in optimized reaction conditions including 22% PEG 8000.

    Article Title: Nucleotide excision repair by dual incisions in plants
    Article Snippet: The method for the isolation and detection of the excised oligonucleotide products of nucleotide excision repair was adapted from previous studies in human cells ( – , , ). .. Arabidopsis cell lysates were incubated with RNase A (1:500; R4642 Sigma) for 10 min at room temperature and then deproteinized by the addition of SDS to a final concentration of 0.33% (from a 10% stock) and incubation with Proteinase K (1:400; P8107S New England Biolabs) for 30 min at 55 °C.

    RNA Extraction:

    Article Title: Freshwater Bacteria Release Methane as a By-Product of Phosphorus Acquisition
    Article Snippet: .. For RNA extraction, cells were stabilized in RNA Protect Reagent (catalog number 76506; Qiagen) and then digested with lysozyme (15 mg ml−1 ) and 20 μl proteinase K (800 U ml−1 , catalog no. P8107S; New England BioLabs) at room temperature for 15 min. RNA was then purified from the bacterial lysate using the Qiagen RNEasy minikit (catalog no. 74104; Qiagen) according to the manufacturer's instructions. .. Genomic DNA contamination was removed by Turbo DNase treatment (catalog no. AM1907; Ambion).

    Article Title: Identification of piRNA binding sites reveals the Argonaute regulatory landscape of the C. elegans germline
    Article Snippet: Paragraph title: Proteinase K treatment and RNA extraction ... Membrane slices were treated with 50 µl of proteinase K (New England Biolabs, P8107S) in proteinase K buffer (100 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM EDTA) for 20 min at 37°C, and then 42% Urea/Proteinase K buffer was added into sample for an additional 20 min at 37°C.

    Article Title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins
    Article Snippet: After one additional wash with high salt wash buffer and two with wash buffer, samples were run on 4–12% NuPAGE Novex Bis-Tris protein gels (ThermoFisher) and transferred to either PVDF (for chemiluminescent imaging) or nitrocellulose (for RNA extraction) membranes. .. A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo).

    Article Title: Characterization of innate immunity genes in the parasitic nematode Brugia malayi
    Article Snippet: Paragraph title: RNA extraction and library preparation ... A further 200 μl of Trizol was used to rinse the pestle in the tube, then 10 μl of proteinase K (Cat. #P8107, New England Biolabs) was added, followed by 30 min of incubation at 55 °C.

    Microscopy:

    Article Title: Kinetic fingerprinting to identify and count single nucleic acids
    Article Snippet: 50 uL of freshly thawed human serum (BioreclamationIVT, #BRH844152) was combined with SDS (final 2% w/v), proteinase K (New England BioLabs, Inc., P8107S; final concentration 0.16 units/μl), and synthetic hsa-miR-141 , and incubated for 15 min at room temperature. .. Next, EDTA was added to a final concentration of 20 mM, and the sample heated to 90 °C in a copper bath for 2 min. After cooling to room temperature for 5 min, each sample was allowed to bind to the microscope coverslip surface for 1 h. Residual serum was removed, the surface washed with 1× PBS, and imaging carried out by objective-type TIRF microscopy as described above.

    Purification:

    Article Title: Freshwater Bacteria Release Methane as a By-Product of Phosphorus Acquisition
    Article Snippet: .. For RNA extraction, cells were stabilized in RNA Protect Reagent (catalog number 76506; Qiagen) and then digested with lysozyme (15 mg ml−1 ) and 20 μl proteinase K (800 U ml−1 , catalog no. P8107S; New England BioLabs) at room temperature for 15 min. RNA was then purified from the bacterial lysate using the Qiagen RNEasy minikit (catalog no. 74104; Qiagen) according to the manufacturer's instructions. .. Genomic DNA contamination was removed by Turbo DNase treatment (catalog no. AM1907; Ambion).

    Article Title: Base-resolution profiling of active DNA demethylation using methylase-assisted bisulfite sequencing (MAB-seq) and caMAB-seq
    Article Snippet: Sodium acetate buffer solution (3 M, pH 5.2±0.1; Sigma-Aldrich, cat. no. S7899) EDTA (0.5 M, pH8.0; Life Technologies, cat. no. 15575-020) EGTA (0.5 M, pH8.0; prepared from Sigma-Aldrich, cat. no. 03777) RNase A (10 mg/mL; Life Technologies, cat. no. EN0531) Anti-H3K4me1 (10 μg/mL; Abcam, cat. no. ab8895) TaqαI (20 U/μL; New England Biolabs, cat. no. R0149L) Klenow fragment (exo– , 5 U/μL; Thermo Scientific, cat. no. EP0422) T4 DNA ligase (2000 U/μL; New England Biolabs, cat. no. M0202M) CutSmart buffer (10×; New England Biolabs, cat. no. B7204S) ATP (100 mM; Thermo Scientific, cat. no. R0441) dNTP set 100 mM solutions (Life Technologies, cat. no. R0181) SPRIselect reagent kit (Beckman Coulter, cat. no. ) DNeasy blood & tissue kit (Qiagen, cat. no. 69504) Qiagen EpiTect DNA bisulfite kit (Qiagen, cat. no. 59104) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) MinElute PCR purification kit (Qiagen, cat. no. 28004) M.SssI (20 U/μL, New England Biolabs, M0226M) NEBuffer 2 (10x; cat. no. B7002S) S-adenosylmethionine (SAM) (32 mM; New England Biolabs, cat. no. B9003S) CRITICAL Always use SAM before its expiration date and make aliquots to avoid multiple cycles of freeze / thaw. .. Unmethylated Lambda DNA (Promega, cat. no. D1521) Agilent High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067-4626) cOmplete EDTA-free proteinase inhibitor (Roche, cat. no. 11873580001) Dynabeads Protein G for Immunoprecipitation (ThermoFisher Scientific, cat. no. 10004D) RNase A (Life Technologies, cat. no. 12091-201) Proteinase K (New England Biolabs, cat. no. P8107S) Glycogen (Roche Life Science, cat. no. 10901393001) 5 PRIME Phase Lock Gel heavy 2 mL (Fisher Scientific, cat. no. FP2302830) Phenol - chloroform - isoamyl alcohol mixture (Sigma-Aldrich, cat. no. 77617) !

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: .. After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions. .. In all, 1/30 of the purified DNA was analyzed by qPCR using the Power SYBR™ Green PCR Master Mix (cat.#4367659, Life Technologies).

    Lysis:

    Article Title: Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm
    Article Snippet: .. Ear clips were lysed overnight at 60°C in a solution with tail lysis buffer (100 mM Tris-HCl pH 8.1, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) supplemented with Proteinase K (NEB, Cat. # P8107S). ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: .. After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions. .. In all, 1/30 of the purified DNA was analyzed by qPCR using the Power SYBR™ Green PCR Master Mix (cat.#4367659, Life Technologies).

    Chromatin Immunoprecipitation:

    Article Title: Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype
    Article Snippet: Paragraph title: Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) ... The eluted chromatin was then subjected to RNase A (EN0531, Thermo Fisher Scientific) and proteinase K (P8107S, NEB) digestion followed by phenol/chloroform extraction of DNA.

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: Paragraph title: Chromatin immunoprecipitation ... After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions.

    Real-time Polymerase Chain Reaction:

    Article Title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins
    Article Snippet: A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo). .. Libraries were PCR amplified with Q5 master mix (NEB) for 6–18 cycles (chosen by performing qPCR on the pre-amplified library).

    Article Title: Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype
    Article Snippet: Paragraph title: Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) ... The eluted chromatin was then subjected to RNase A (EN0531, Thermo Fisher Scientific) and proteinase K (P8107S, NEB) digestion followed by phenol/chloroform extraction of DNA.

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions. .. In all, 1/30 of the purified DNA was analyzed by qPCR using the Power SYBR™ Green PCR Master Mix (cat.#4367659, Life Technologies).

    Multiplex Assay:

    Article Title: Base-resolution profiling of active DNA demethylation using methylase-assisted bisulfite sequencing (MAB-seq) and caMAB-seq
    Article Snippet: KAPA HiFi Uracil+ HotStart ReadyMix (Kapa Biosystems, KK2801) NEBNext Multiplex Oligos for Illumina (Index Primers Set 1; New England Biolabs, cat. no. E7335L) NEBNext Multiplex Oligos for Illumina (Index Primers Set 2; New England Biolabs, cat. no. E7500L) NEBNext ultra DNA Library Prep Kit for Illumina (New England Biolabs, cat. no. E7370S) NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs, cat. no. E6040S) Custom methylated adapters (asterisk denotes phosphorothioate bond, all cytosines are modified as 5mC; Integrated DNA Technologies): Forward: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ Reverse: 5′-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3′ Custom 5hmC/5fC/5caC-modified oligo (X: 5hmC, 5fC or 5caC): Forward: 5′-AGCCXGXGCXGXGCXGGTXGAGXGGCXGCTCCXGCAGC-3′, Reverse: 5′-GCTGXGGGAGXGGCXGCTXGACXGGXGXGGXGXGGGCT-3′ CRITICAL Only CpG site of these oligos are modified, which is different from the methylated adaptor in which all cytosines are modified as 5mC. .. Unmethylated Lambda DNA (Promega, cat. no. D1521) Agilent High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067-4626) cOmplete EDTA-free proteinase inhibitor (Roche, cat. no. 11873580001) Dynabeads Protein G for Immunoprecipitation (ThermoFisher Scientific, cat. no. 10004D) RNase A (Life Technologies, cat. no. 12091-201) Proteinase K (New England Biolabs, cat. no. P8107S) Glycogen (Roche Life Science, cat. no. 10901393001) 5 PRIME Phase Lock Gel heavy 2 mL (Fisher Scientific, cat. no. FP2302830) Phenol - chloroform - isoamyl alcohol mixture (Sigma-Aldrich, cat. no. 77617) !

    Agarose Gel Electrophoresis:

    Article Title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins
    Article Snippet: A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo). .. The 175–300nt fragment was size-selected by agarose gel electrophoresis and gel extracted (MinElute Gel Extraction, Qiagen).

    Ethanol Precipitation:

    Article Title: Nucleotide excision repair by dual incisions in plants
    Article Snippet: Arabidopsis cell lysates were incubated with RNase A (1:500; R4642 Sigma) for 10 min at room temperature and then deproteinized by the addition of SDS to a final concentration of 0.33% (from a 10% stock) and incubation with Proteinase K (1:400; P8107S New England Biolabs) for 30 min at 55 °C. .. Following phenol chloroform extraction and ethanol precipitation, DNA samples were resuspended in 10 μL of water.

    Produced:

    Article Title: Base-resolution profiling of active DNA demethylation using methylase-assisted bisulfite sequencing (MAB-seq) and caMAB-seq
    Article Snippet: Hydrogen gas is produced when NaBH4 reacts with water. .. Unmethylated Lambda DNA (Promega, cat. no. D1521) Agilent High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067-4626) cOmplete EDTA-free proteinase inhibitor (Roche, cat. no. 11873580001) Dynabeads Protein G for Immunoprecipitation (ThermoFisher Scientific, cat. no. 10004D) RNase A (Life Technologies, cat. no. 12091-201) Proteinase K (New England Biolabs, cat. no. P8107S) Glycogen (Roche Life Science, cat. no. 10901393001) 5 PRIME Phase Lock Gel heavy 2 mL (Fisher Scientific, cat. no. FP2302830) Phenol - chloroform - isoamyl alcohol mixture (Sigma-Aldrich, cat. no. 77617) !

    Immunoprecipitation:

    Article Title: CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins
    Article Snippet: For TAG-eCLIP experiments, all western blot imaging to validate successful immunoprecipitation was done using primary antibody against the native protein (including immunoprecipitations with anti-tag antibodies). .. A range from protein size to 75 kDa above protein size was isolated, incubated first for 20 minutes at 37°C with 200 μL PK buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA plus 40 μL Proteinase K (NEB P8107S)), followed by 20 minutes at 37°C with 200 μL PK-Urea buffer (160 μL of 100 mM TrisHCl, pH 7.4, 50 mM NaCl, 10 mM EDTA, 7M Urea plus 40 μL Proteinase K (NEB P8107S), after which RNA was isolated using phenol-chloroform extraction followed by RNA Clean & Concentrator column cleanup (Zymo).

    Article Title: Base-resolution profiling of active DNA demethylation using methylase-assisted bisulfite sequencing (MAB-seq) and caMAB-seq
    Article Snippet: .. Unmethylated Lambda DNA (Promega, cat. no. D1521) Agilent High Sensitivity DNA Kit (Agilent Technologies, cat. no. 5067-4626) cOmplete EDTA-free proteinase inhibitor (Roche, cat. no. 11873580001) Dynabeads Protein G for Immunoprecipitation (ThermoFisher Scientific, cat. no. 10004D) RNase A (Life Technologies, cat. no. 12091-201) Proteinase K (New England Biolabs, cat. no. P8107S) Glycogen (Roche Life Science, cat. no. 10901393001) 5 PRIME Phase Lock Gel heavy 2 mL (Fisher Scientific, cat. no. FP2302830) Phenol - chloroform - isoamyl alcohol mixture (Sigma-Aldrich, cat. no. 77617) ! ..

    Article Title: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration
    Article Snippet: .. After 0.2 mg/ml Proteinase K (cat.#P8107S, New England BioLabs) treatment of samples, DNA from immunoprecipitated samples as well as DNA from 10% input was purified by phenol and chloroform extraction and using the QIAquick PCR Purification Kit (cat.#28106, Qiagen) following the manufacturer’s instructions. .. In all, 1/30 of the purified DNA was analyzed by qPCR using the Power SYBR™ Green PCR Master Mix (cat.#4367659, Life Technologies).

    Staining:

    Article Title: Healing of Preterm Ruptured Fetal Membranes
    Article Snippet: Paragraph title: Immunofluorescent staining ... After sections (5 μm) were deparaffinized, antigen retrieval was performed by incubation with proteinase K (P8107S, New England Biolab, working concentration, 0.6 units/ml) for 1 minute at 37 °C.

    Cross-linking Immunoprecipitation:

    Article Title: Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm
    Article Snippet: Genomic DNA from a right ear clip was used for PCR genotyping. .. Ear clips were lysed overnight at 60°C in a solution with tail lysis buffer (100 mM Tris-HCl pH 8.1, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) supplemented with Proteinase K (NEB, Cat. # P8107S).

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    New England Biolabs p8107s
    P8107s, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pngase f
    Pngase F, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pngase f/product/New England Biolabs
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    Thermolabile Proteinase K is an engineered subtilisin related serine protease that cleaves the peptide bond at the carboxyl side of aliphatic or aromatic amino acid residues and will hydrolyze a
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