tris hcl buffer  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    Magnesium chloride
    Description:

    Catalog Number:
    m4880
    Price:
    None
    Buy from Supplier


    Structured Review

    Millipore tris hcl buffer
    Magnesium chloride

    https://www.bioz.com/result/tris hcl buffer/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris hcl buffer - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function"

    Article Title: Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153798

    Activity of protein preparations on pre-tRNA at low ammonium acetate. The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.
    Figure Legend Snippet: Activity of protein preparations on pre-tRNA at low ammonium acetate. The pre-tRNA processing activity with different amounts of RNase P protein preparations alone, in the absence of RNA component, was assayed in 50 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride and 100 mM ammonium acetate. S denotes the substrate alone reaction.

    Techniques Used: Activity Assay

    Related Articles

    other:

    Article Title: In vitro evaluation of the toxic effects of MgO nanostructure in Hela cell line
    Article Snippet: Chemicals and Synthesis of MgO Nanotubes Magnesium chloride (MgCl2 ), sodium hydroxide (NaOH), polyvinyl alcohol (PVA), and ethanol were purchased from Sigma Aldrich and used as chemical reagents.

    Incubation:

    Article Title: Inducible dissociation of SCFMet30 ubiquitin ligase mediates a rapid transcriptional response to cadmium
    Article Snippet: Recombinant His6-HA Met4 produced in insect cells was affinity purified on nickel resin and treated with lambda phosphatase. .. Immobilized SCFMet30 was washed three times with 1 ml of ubiquitination buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 2 mM ATP, 50 μM DTT), incubated with His6 Uba1 (E1), His6 Cdc34 (E2), ubiquitin (Sigma) or methylated ubiquitin (Affiniti) and Met4 in 10 μl ubiquitylation buffer at 30°C for 1 h. Products were separated by SDS–PAGE and immunoblotted with anti-HA (12CA5) antibody. ..

    Article Title: Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization
    Article Snippet: .. The plates were further incubated for 30 min, the cell monolayers were washed two times with sterile PBS at room temperature and then 100 μl tubulin extraction buffer (1 mM MgCl2 , 2 mM EGTA, 0.5% NP40 and 20 mM Tris-HCl (pH 6.8)) supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich; cat. no. P8340) was added per well. .. After a short and vigorous vortex mixing, the cell lysates were incubated at room temperature for 5 min and then centrifuged at 16 000 r.p.m. for 10 min to separate the soluble and polymerized tubulin fractions.

    Methylation:

    Article Title: Inducible dissociation of SCFMet30 ubiquitin ligase mediates a rapid transcriptional response to cadmium
    Article Snippet: Recombinant His6-HA Met4 produced in insect cells was affinity purified on nickel resin and treated with lambda phosphatase. .. Immobilized SCFMet30 was washed three times with 1 ml of ubiquitination buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 2 mM ATP, 50 μM DTT), incubated with His6 Uba1 (E1), His6 Cdc34 (E2), ubiquitin (Sigma) or methylated ubiquitin (Affiniti) and Met4 in 10 μl ubiquitylation buffer at 30°C for 1 h. Products were separated by SDS–PAGE and immunoblotted with anti-HA (12CA5) antibody. ..

    SDS Page:

    Article Title: Inducible dissociation of SCFMet30 ubiquitin ligase mediates a rapid transcriptional response to cadmium
    Article Snippet: Recombinant His6-HA Met4 produced in insect cells was affinity purified on nickel resin and treated with lambda phosphatase. .. Immobilized SCFMet30 was washed three times with 1 ml of ubiquitination buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 2 mM ATP, 50 μM DTT), incubated with His6 Uba1 (E1), His6 Cdc34 (E2), ubiquitin (Sigma) or methylated ubiquitin (Affiniti) and Met4 in 10 μl ubiquitylation buffer at 30°C for 1 h. Products were separated by SDS–PAGE and immunoblotted with anti-HA (12CA5) antibody. ..

    Recombinant:

    Article Title: Targeting of Dicer-2 and RNA by a Viral RNA Silencing Suppressor in Drosophila Cells
    Article Snippet: RNase III-mediated cleavage assays were conducted according to our standard protocol , using T7-transcribed 500-bp dsRNA derived from GFP as precursor. .. Reactions were performed in 20-μl reaction mixture volumes containing 0.1 μM dsRNA, 1× RNase III buffer (20 mM Tris-HCl, 0.5 mM EDTA, 5 mM MgCl2 , 1 mM DTT [Sigma, SaintLouis, MO], 140 mM NaCl, 2.7 mM KCl [pH 7.9]), and 1 U recombinant RNase III (a generous gift from Yi Zhang). .. After incubation at 37°C for 30 min, reaction products were resolved by Tris-borate-EDTA (TBE)–agarose gel electrophoresis on 1.2% gels, and the RNA was stained with ethidium bromide at 1 μg/ml.

    Activity Assay:

    Article Title: Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures
    Article Snippet: .. Oxygen evolution activity of the preparations was determined using a Clark-type electrode (Hansatech Instruments, UK) with 10 μg chl of the isolated thylakoid preparations in buffer B (50mM MES/NaOH pH = 6.0, 15mM NaCl, 5mM MgCl, 2mM CaCl2 ) and 0.3 mM 2,6-dichlorobenzoquinone (DCBQ, Sigma) as the exogenous electron acceptor. ..

    Isolation:

    Article Title: Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures
    Article Snippet: .. Oxygen evolution activity of the preparations was determined using a Clark-type electrode (Hansatech Instruments, UK) with 10 μg chl of the isolated thylakoid preparations in buffer B (50mM MES/NaOH pH = 6.0, 15mM NaCl, 5mM MgCl, 2mM CaCl2 ) and 0.3 mM 2,6-dichlorobenzoquinone (DCBQ, Sigma) as the exogenous electron acceptor. ..

    Protease Inhibitor:

    Article Title: Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization
    Article Snippet: .. The plates were further incubated for 30 min, the cell monolayers were washed two times with sterile PBS at room temperature and then 100 μl tubulin extraction buffer (1 mM MgCl2 , 2 mM EGTA, 0.5% NP40 and 20 mM Tris-HCl (pH 6.8)) supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich; cat. no. P8340) was added per well. .. After a short and vigorous vortex mixing, the cell lysates were incubated at room temperature for 5 min and then centrifuged at 16 000 r.p.m. for 10 min to separate the soluble and polymerized tubulin fractions.

    Crystallization Assay:

    Article Title: Structural basis of Mycobacterium tuberculosistranscription and transcription inhibition
    Article Snippet: For Mtb RPitc2, the complex for crystallization was prepared by mixing 16 μl 50 μM Mtb RNAP holoenzyme (in 20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM dithiothreitol, 5 mM MgCl2 ), 4 μl 0.4 mM of nucleic-acid scaffold RPo (in 5 mM Tris-HCl, pH 7.7, 0.2 M NaCl, and 10 mM MgCl2 ), and 1 μl 25 mM GpA (in water), and incubating 1 h at 25°C. .. For Mtb RPo, RPitc3, and RPitc4 in complex with Rif, complexes for crystallization were prepared by incubating 16 μl 50 μM Mtb RNAP σA holoenzyme (in 20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM MgCl2 , and 5 mM dithiothreitol,) and 0.5 μl 8 mM Rif (Sigma-Aldrich, Inc.), 0.5 h at 25°C, and then adding 4 μl 0.4 mM of nucleic-acid scaffold RPo, RPitc3, or RPitc4 (in 5 mM Tris-HCl, pH 7.7, 0.2 M NaCl, and 10 mM MgCl2 ), and incubating 1 h at 25°C. .. For Mtb RPitc,2 in complex with Rif, the complex for crystallization was prepared by incubating 16 μl 50 μM Mtb RNAP σA holoenzyme (in 20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM MgCl2 , and 5 mM dithiothreitol) and 0.5 μl 8 mM Rif, 0.5 h at 25°C, and then adding 4 μl 0.4 mM of nucleic-acid scaffold RPo (in 5 mM Tris-HCl, pH 7.7, 0.2 M NaCl, and 10 mM MgCl2 ) and 1 μl 25 mM GpA (in water) and incubating 1 h at 25°C.

    Immunoprecipitation:

    Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3
    Article Snippet: For chain cleavage assays, DUBs (30 ng μl−1 ) were incubated in DUB buffer (50 mM Tris–HCl pH 7.5, 5 mM DTT, 100 mM NaCl) with the indicated ubiquitin chains (0.13 μg μl−1 , K48 ub2–7 , K63 ub2–7 (Boston Biochem), K48 ub2 (Axel Knebel)) on an IP shaker for 1 h at 30 °C. .. Ubiquitylation assays of SMAD2 or FLAG-SMAD2/3/4 (immunoprecipitated from HEK293 cells treated with 50 pM TGFβ for 1 h before lysis) were performed in ubiquitylation assay buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 2 μM ATP) with His6 -UBE1 (0.1 μM), UBE2D1 (1 μM or indicated concentrations), His6 -NEDD4L (1 μM) and ubiquitin (50 μM; Sigma) for 1 h at 30 °C on an IP shaker. ..

    Lysis:

    Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3
    Article Snippet: For chain cleavage assays, DUBs (30 ng μl−1 ) were incubated in DUB buffer (50 mM Tris–HCl pH 7.5, 5 mM DTT, 100 mM NaCl) with the indicated ubiquitin chains (0.13 μg μl−1 , K48 ub2–7 , K63 ub2–7 (Boston Biochem), K48 ub2 (Axel Knebel)) on an IP shaker for 1 h at 30 °C. .. Ubiquitylation assays of SMAD2 or FLAG-SMAD2/3/4 (immunoprecipitated from HEK293 cells treated with 50 pM TGFβ for 1 h before lysis) were performed in ubiquitylation assay buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 2 μM ATP) with His6 -UBE1 (0.1 μM), UBE2D1 (1 μM or indicated concentrations), His6 -NEDD4L (1 μM) and ubiquitin (50 μM; Sigma) for 1 h at 30 °C on an IP shaker. ..

    Ubiquitin Assay:

    Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3
    Article Snippet: For chain cleavage assays, DUBs (30 ng μl−1 ) were incubated in DUB buffer (50 mM Tris–HCl pH 7.5, 5 mM DTT, 100 mM NaCl) with the indicated ubiquitin chains (0.13 μg μl−1 , K48 ub2–7 , K63 ub2–7 (Boston Biochem), K48 ub2 (Axel Knebel)) on an IP shaker for 1 h at 30 °C. .. Ubiquitylation assays of SMAD2 or FLAG-SMAD2/3/4 (immunoprecipitated from HEK293 cells treated with 50 pM TGFβ for 1 h before lysis) were performed in ubiquitylation assay buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 2 μM ATP) with His6 -UBE1 (0.1 μM), UBE2D1 (1 μM or indicated concentrations), His6 -NEDD4L (1 μM) and ubiquitin (50 μM; Sigma) for 1 h at 30 °C on an IP shaker. ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Millipore pfo running buffer
    <t>PFO-PAGE</t> reveals PFO maintains claudin-4 oligomers. ( A ) Cldn-4 SF9 membranes were solubilized for 1 h at RT in PFO, spun 30 min at 100,000 g and the resultant high speed (HS) supernatant was mixed with PFO sample buffer. Sample was loaded on 4%–20% native gels <t>preelectrophoresed</t> in PFO-containing running buffer. Anti-His immunoblot of HS sup (lanes 1,2 ) or PFO-purified protein (lane 3 ) reveals a range of sizes (long exposure), with the majority of protein concentrated at ∼120 kD (short exposure and purified protein). ( B ) The distance migrated by soluble protein standards versus their log MW is plotted, showing a linear relationship between molecular mass and migration in the PFO-PAGE system.
    Pfo Running Buffer, supplied by Millipore, 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/pfo running buffer/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pfo running buffer - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

    99
    Millipore immunoprecipitation buffer
    ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z <t>immunoprecipitation.</t> Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.
    Immunoprecipitation Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    immunoprecipitation buffer - by Bioz Stars, 2021-03
    99/100 stars
      Buy from Supplier

    99
    Millipore parp1 reaction buffer
    Mutational Analysis of the DNA-Binding Site (A) Solvent-accessible surface of the DNA-binding site colored by electrostatic potential (calculated in PyMol). Residues perturbed by DNA binding (including Arg399 and Arg400) map to an intensely positively charged surface patch (left), whose polarity is predicted to be reversed by the combination of R399D and R400Q mutations (right). ) of XRCC1-BRCT1 variants and mutants. Both codon 399 variants and the putative DNA binding disruptive R399D,R400Q double mutant bind tightly to PAR chains generated on plates coated with histone H1 and incubated with <t>PARP1</t> and NAD + ). No binding is seen for any of the constructs in the absence of NAD + . Data represent the mean of four measurements of three separate replicates analyzed by two-way ANOVA. Error bars show ± 1 SEM. (C) Fluorescence polarization assays of XRCC1-BRCT1 variants and mutants to FITC-labeled nicked dsDNA oligonucleotides with (left) or without (right) 5′ phosphorylation at the nick site. The codon 399 variants and the PAR-binding site mutant all bind with high affinity to both nicked duplex oligonucleotides, whereas the R399D,R400Q double mutant shows very low fluorescence poloarization (FP) values, which cannot be fitted to a binding curve (for K d B). Data represent the mean of four measurements comprised of two separate replicates with XRCC1-BRCT1 from two separate protein purifications. Error bars show ± 1 SEM.
    Parp1 Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/parp1 reaction buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    parp1 reaction buffer - by Bioz Stars, 2021-03
    99/100 stars
      Buy from Supplier

    97
    Millipore t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by Millipore, 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/t4 rna ligase buffer/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

    Image Search Results


    PFO-PAGE reveals PFO maintains claudin-4 oligomers. ( A ) Cldn-4 SF9 membranes were solubilized for 1 h at RT in PFO, spun 30 min at 100,000 g and the resultant high speed (HS) supernatant was mixed with PFO sample buffer. Sample was loaded on 4%–20% native gels preelectrophoresed in PFO-containing running buffer. Anti-His immunoblot of HS sup (lanes 1,2 ) or PFO-purified protein (lane 3 ) reveals a range of sizes (long exposure), with the majority of protein concentrated at ∼120 kD (short exposure and purified protein). ( B ) The distance migrated by soluble protein standards versus their log MW is plotted, showing a linear relationship between molecular mass and migration in the PFO-PAGE system.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Expression, solubilization, and biochemical characterization of the tight junction transmembrane protein claudin-4

    doi:

    Figure Lengend Snippet: PFO-PAGE reveals PFO maintains claudin-4 oligomers. ( A ) Cldn-4 SF9 membranes were solubilized for 1 h at RT in PFO, spun 30 min at 100,000 g and the resultant high speed (HS) supernatant was mixed with PFO sample buffer. Sample was loaded on 4%–20% native gels preelectrophoresed in PFO-containing running buffer. Anti-His immunoblot of HS sup (lanes 1,2 ) or PFO-purified protein (lane 3 ) reveals a range of sizes (long exposure), with the majority of protein concentrated at ∼120 kD (short exposure and purified protein). ( B ) The distance migrated by soluble protein standards versus their log MW is plotted, showing a linear relationship between molecular mass and migration in the PFO-PAGE system.

    Article Snippet: Briefly, 4%–20% native gradient gels (Jule Biotechnologies or Bio-Rad) were preelectrophoresed in PFO-running buffer (25 mM Tris base, 192 mM glycine, 0.5% (w/v) PFO (Aldrich), pH of buffer 8.0) ( ) for 45 min at 50 volts.

    Techniques: Polyacrylamide Gel Electrophoresis, Purification, Migration

    ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Journal: Genes & Development

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    doi: 10.1101/gad.1547707

    Figure Lengend Snippet: ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Article Snippet: Mononucleosomes (0.7 mL) in immunoprecipitation buffer (10 mM Tris-HCl at pH 7.4, 50 mM or 10 mM NaCl, 0.2 mM EDTA) were incubated with 100 μL of anti-Flag M2 affinity gel overnight at 4°C (Sigma) and then washed five times with immunoprecipitation buffer.

    Techniques: Chromatin Immunoprecipitation, Expressing, Immunoprecipitation

    Mutational Analysis of the DNA-Binding Site (A) Solvent-accessible surface of the DNA-binding site colored by electrostatic potential (calculated in PyMol). Residues perturbed by DNA binding (including Arg399 and Arg400) map to an intensely positively charged surface patch (left), whose polarity is predicted to be reversed by the combination of R399D and R400Q mutations (right). ) of XRCC1-BRCT1 variants and mutants. Both codon 399 variants and the putative DNA binding disruptive R399D,R400Q double mutant bind tightly to PAR chains generated on plates coated with histone H1 and incubated with PARP1 and NAD + ). No binding is seen for any of the constructs in the absence of NAD + . Data represent the mean of four measurements of three separate replicates analyzed by two-way ANOVA. Error bars show ± 1 SEM. (C) Fluorescence polarization assays of XRCC1-BRCT1 variants and mutants to FITC-labeled nicked dsDNA oligonucleotides with (left) or without (right) 5′ phosphorylation at the nick site. The codon 399 variants and the PAR-binding site mutant all bind with high affinity to both nicked duplex oligonucleotides, whereas the R399D,R400Q double mutant shows very low fluorescence poloarization (FP) values, which cannot be fitted to a binding curve (for K d B). Data represent the mean of four measurements comprised of two separate replicates with XRCC1-BRCT1 from two separate protein purifications. Error bars show ± 1 SEM.

    Journal: Cell Reports

    Article Title: Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1

    doi: 10.1016/j.celrep.2018.12.082

    Figure Lengend Snippet: Mutational Analysis of the DNA-Binding Site (A) Solvent-accessible surface of the DNA-binding site colored by electrostatic potential (calculated in PyMol). Residues perturbed by DNA binding (including Arg399 and Arg400) map to an intensely positively charged surface patch (left), whose polarity is predicted to be reversed by the combination of R399D and R400Q mutations (right). ) of XRCC1-BRCT1 variants and mutants. Both codon 399 variants and the putative DNA binding disruptive R399D,R400Q double mutant bind tightly to PAR chains generated on plates coated with histone H1 and incubated with PARP1 and NAD + ). No binding is seen for any of the constructs in the absence of NAD + . Data represent the mean of four measurements of three separate replicates analyzed by two-way ANOVA. Error bars show ± 1 SEM. (C) Fluorescence polarization assays of XRCC1-BRCT1 variants and mutants to FITC-labeled nicked dsDNA oligonucleotides with (left) or without (right) 5′ phosphorylation at the nick site. The codon 399 variants and the PAR-binding site mutant all bind with high affinity to both nicked duplex oligonucleotides, whereas the R399D,R400Q double mutant shows very low fluorescence poloarization (FP) values, which cannot be fitted to a binding curve (for K d B). Data represent the mean of four measurements comprised of two separate replicates with XRCC1-BRCT1 from two separate protein purifications. Error bars show ± 1 SEM.

    Article Snippet: The adsorbed proteins were mock ribosylated in the absence of NAD+ or ribosylated in the presence of the 50 mM NAD+ (Sigma) in PARP1 reaction buffer (50 mM Tris–HCl pH7.5, 0.8 mM MgCl2 , 1% glycerol and 1.5 mM DTT) containing 40 nM single-stranded oligodeoxyribonucleotide (5′-CATATGCCGGAGATCCGCCTCC-3′) and 5 nM human PARP1 (Trevigen) in a final volume of 50 μL at room temp for 30 min. After rinsing (4 × ) with 50 μL of 0.1% Tween 20 in PBS, 50 μL of His-SUMO-XRCC-BRCT1 or its variants (diluted to 25 nM in 20 mM Tris pH7.5, 130 nM NaCl) were added to the adsorbed proteins and incubated on ice for 30 min.

    Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Construct, Fluorescence, Labeling

    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: The reaction was set up as follows: 5.4 μL of sample, 0.6 μL of 3′ adapter (100 μM), 1 μL of 10× T4 RNA ligase buffer (50 mM Tris-HCl at pH7.8, 10 mM MgCl2 , 1 mM ATP, 10 mM DTT), 1 μL of DMSO (Sigma), 1 μL of RNase out (Invitrogen), 20 U of T4 RNA ligase 1 (Invitrogen).

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing