histone h2b human recombinant  (New England Biolabs)


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    Name:
    Histone H2B Human Recombinant
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    Histone H2B Human Recombinant 100 ug
    Catalog Number:
    m2505s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
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    New England Biolabs histone h2b human recombinant
    Histone H2B Human Recombinant
    Histone H2B Human Recombinant 100 ug
    https://www.bioz.com/result/histone h2b human recombinant/product/New England Biolabs
    Average 93 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    histone h2b human recombinant - by Bioz Stars, 2020-08
    93/100 stars

    Images

    1) Product Images from "Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming \u03c9-NG-Monomethylated Arginine Residues"

    Article Title: Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming \u03c9-NG-Monomethylated Arginine Residues

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.336271

    GST-PRMT7 can methylate histone H2B producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μ m ) with histone H2B (10 μg; 12.1 μ m ) was carried out for 1 (■), 5 (♦), and 25 h (●)
    Figure Legend Snippet: GST-PRMT7 can methylate histone H2B producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μ m ) with histone H2B (10 μg; 12.1 μ m ) was carried out for 1 (■), 5 (♦), and 25 h (●)

    Techniques Used: In Vitro, Methylation

    2) Product Images from "APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells "

    Article Title: APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.02243-07

    The tandem zinc finger domain of APLF binds poly(ADP-ribosyl)ated proteins in vitro. (A) Recombinant human His-APLF proteins. One microgram of recombinant His-APLF and the indicated mutated derivatives were fractionated by SDS-PAGE and stained with Coomassie blue. A schematic of the recombinant His-APLF proteins is shown (bottom). “H-” and “FHA” denote the decahistidine tag and FHA domain, respectively. The C-terminal acidic tail (gray box) and tandem ZNF domains (black boxes: *, ZNF1; **, ZNF2) are also indicated. Dotted boxes denote a mutated ZNF. Amino acid positions are indicated at the top. (B) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated (pADPr-h1) or not (h1) as described above and incubated in the absence (−) or presence of 17.5 nM of the indicated His-APLF protein for 30 min on ice. Bound His-APLF was quantified as described above. (C) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated and incubated with the indicated concentration of the indicated His-APLF protein for 30 min at 37°C. Bound His-APLF was quantified as described above. (D) Two micrograms of BSA, calf thymus histone H1, recombinant human histone H2B, or recombinant human PARP-1 was slot blotted onto nitrocellulose and mock treated (−pADPr) or poly(ADP-ribosyl)ated (+pADPr) with recombinant human PARP-1 (P1). Filters were incubated with 35 nM His-APLF, and after extensive washing, filter-bound APLF was detected with anti-His-Tag MAb and appropriate secondary antibody. Error bars show standard errors of the mean.
    Figure Legend Snippet: The tandem zinc finger domain of APLF binds poly(ADP-ribosyl)ated proteins in vitro. (A) Recombinant human His-APLF proteins. One microgram of recombinant His-APLF and the indicated mutated derivatives were fractionated by SDS-PAGE and stained with Coomassie blue. A schematic of the recombinant His-APLF proteins is shown (bottom). “H-” and “FHA” denote the decahistidine tag and FHA domain, respectively. The C-terminal acidic tail (gray box) and tandem ZNF domains (black boxes: *, ZNF1; **, ZNF2) are also indicated. Dotted boxes denote a mutated ZNF. Amino acid positions are indicated at the top. (B) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated (pADPr-h1) or not (h1) as described above and incubated in the absence (−) or presence of 17.5 nM of the indicated His-APLF protein for 30 min on ice. Bound His-APLF was quantified as described above. (C) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated and incubated with the indicated concentration of the indicated His-APLF protein for 30 min at 37°C. Bound His-APLF was quantified as described above. (D) Two micrograms of BSA, calf thymus histone H1, recombinant human histone H2B, or recombinant human PARP-1 was slot blotted onto nitrocellulose and mock treated (−pADPr) or poly(ADP-ribosyl)ated (+pADPr) with recombinant human PARP-1 (P1). Filters were incubated with 35 nM His-APLF, and after extensive washing, filter-bound APLF was detected with anti-His-Tag MAb and appropriate secondary antibody. Error bars show standard errors of the mean.

    Techniques Used: In Vitro, Recombinant, SDS Page, Staining, Incubation, Concentration Assay

    3) Product Images from "Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region"

    Article Title: Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region

    Journal: Biochemistry

    doi: 10.1021/bi800309m

    Kinetic analysis of MST1 protein constructs. (A) Lineweaver–Burk plot comparing the activity of MST1-FL and the kinase domain (MST1-KD) toward FoxO1. (B) Lineweaver–Burk plot depicting the activity of MST-KD toward histone H2B. (C) Lineweaver–Burk plot depicting the activity of MST1-FL toward histone H2B. (D) Summary of the various steady state parameters for the plots shown in panels A–C. (E and F) Autoradiography gel showing the activity of MST1-FL and MST1-KD toward FoxO1 and histone H2B, respectively. The gel shows the incorporation of the γ- 32 P label as a function of substrate concentration. All reactions were carried out for 15 min and then stopped with the addition of SDS–PAGE loading buffer.
    Figure Legend Snippet: Kinetic analysis of MST1 protein constructs. (A) Lineweaver–Burk plot comparing the activity of MST1-FL and the kinase domain (MST1-KD) toward FoxO1. (B) Lineweaver–Burk plot depicting the activity of MST-KD toward histone H2B. (C) Lineweaver–Burk plot depicting the activity of MST1-FL toward histone H2B. (D) Summary of the various steady state parameters for the plots shown in panels A–C. (E and F) Autoradiography gel showing the activity of MST1-FL and MST1-KD toward FoxO1 and histone H2B, respectively. The gel shows the incorporation of the γ- 32 P label as a function of substrate concentration. All reactions were carried out for 15 min and then stopped with the addition of SDS–PAGE loading buffer.

    Techniques Used: Construct, Activity Assay, Microscale Thermophoresis, Autoradiography, Concentration Assay, SDS Page

    MST1 preparation and characterization. (A) The construct designs for the various protein fragments of MST1 used for this study are depicted. The various domains are abbreviated as follows: KD, kinase domain; RR, regulatory region; and DD, dimerization domain. (B) SDS page gel of the various recombinant protein constructs used in this study. The abbreviations are as follows: MST1-FL, full-length MST1; MST1-KD/RR, MST1 kinase with the regulatory region, spanning residues 1–380; and MST1-FL (TM), MST1-FL triple mutant with residues S340, T346, and T348 mutated to alanine. (C) Autoradiography gel showing the activity of MST1 toward phosphorylation of its substrates, FoxO1 and histone H2B. The signal depicted is the incorporation of the γ- 32 P label onto FoxO and histone H2B. The autophosphorylation signal of MST1 is also visible as indicated. (D) Autoradiograph showing the effect of DNA binding on the phosphorylation activity of FoxO1 by MST1-FL in the presence and absence of its cognate DNA, the Daf-16 binding element
    Figure Legend Snippet: MST1 preparation and characterization. (A) The construct designs for the various protein fragments of MST1 used for this study are depicted. The various domains are abbreviated as follows: KD, kinase domain; RR, regulatory region; and DD, dimerization domain. (B) SDS page gel of the various recombinant protein constructs used in this study. The abbreviations are as follows: MST1-FL, full-length MST1; MST1-KD/RR, MST1 kinase with the regulatory region, spanning residues 1–380; and MST1-FL (TM), MST1-FL triple mutant with residues S340, T346, and T348 mutated to alanine. (C) Autoradiography gel showing the activity of MST1 toward phosphorylation of its substrates, FoxO1 and histone H2B. The signal depicted is the incorporation of the γ- 32 P label onto FoxO and histone H2B. The autophosphorylation signal of MST1 is also visible as indicated. (D) Autoradiograph showing the effect of DNA binding on the phosphorylation activity of FoxO1 by MST1-FL in the presence and absence of its cognate DNA, the Daf-16 binding element

    Techniques Used: Construct, SDS Page, Recombinant, Mutagenesis, Autoradiography, Activity Assay, Binding Assay

    4) Product Images from "Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia"

    Article Title: Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia

    Journal: F1000Research

    doi: 10.12688/f1000research.2-148.v1

    Linker histone H1 is neurotoxic. Dissociated cortical neurons were cultured for 48 hrs and histones were applied for a further 24 hrs. Cell death was determined by luminescence to measure protease release and was expressed as relative luminescence units (RLU). For clarity RLU means are expressed as +/- 2 x standard deviation (SD). Histone H1 caused dose-dependent death of cortical neurons ( a ) while core histones H2A, H2B, H3 and H4 were without effect up to 200 nM ( b–e ). Cell death as measured by luminescence was confirmed directly via phase contrast microscopy of dissociated cortical neurons in culture ( Figure 4 ).
    Figure Legend Snippet: Linker histone H1 is neurotoxic. Dissociated cortical neurons were cultured for 48 hrs and histones were applied for a further 24 hrs. Cell death was determined by luminescence to measure protease release and was expressed as relative luminescence units (RLU). For clarity RLU means are expressed as +/- 2 x standard deviation (SD). Histone H1 caused dose-dependent death of cortical neurons ( a ) while core histones H2A, H2B, H3 and H4 were without effect up to 200 nM ( b–e ). Cell death as measured by luminescence was confirmed directly via phase contrast microscopy of dissociated cortical neurons in culture ( Figure 4 ).

    Techniques Used: Cell Culture, Standard Deviation, Microscopy

    Release of histones H1 and H2B from ischaemic adult brain. Adult conditioned medium (ACM) was made by incubating ischaemic brain slices in defined culture medium that was subjected to affinity chromatography. Western blots using antibodies specific to histones H1 and H2B show the presence of histone H1 at 32 kDa (lane 1) and histone H2B at 16 kDa in ACM (lane 3). Protein standards are shown at 32 kDa (H1 lane 2) and at 16 kDa for the monomer and 32 kDa for the dimer (H2B lane 4).
    Figure Legend Snippet: Release of histones H1 and H2B from ischaemic adult brain. Adult conditioned medium (ACM) was made by incubating ischaemic brain slices in defined culture medium that was subjected to affinity chromatography. Western blots using antibodies specific to histones H1 and H2B show the presence of histone H1 at 32 kDa (lane 1) and histone H2B at 16 kDa in ACM (lane 3). Protein standards are shown at 32 kDa (H1 lane 2) and at 16 kDa for the monomer and 32 kDa for the dimer (H2B lane 4).

    Techniques Used: Affinity Chromatography, Western Blot

    5) Product Images from "Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies"

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20161190

    Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.
    Figure Legend Snippet: Histone H2A-reactive IgM monoclonal antibodies, isolated from B6.Sle123 mice, neutralize tier 2 strains of HIV-1. Hybridomas were generated from splenocytes isolated from B6.Sle123 mice that displayed serum tier 2 HIV-1 neutralization ( n = 2). (A) Purified monoclonal IgM and IgG antibodies ( n = 8) were tested for HIV-1 neutralization against 4 strains of HIV-1 and reported as IC 50 (the concentration of antibody required for 50% neutralization). (B) Specificities of the monoclonal antibodies were tested using ELISA against the histone H2A, histone H2B, and chromatin nuclear antigens in addition to HIV-1 gp140 (YU2) Env and the CD4bs (RSC3) epitope, as well as a CD4bs-negative control (ΔRSC3). The two neutralizing IgM mAbs (P4E4 and O4C5) are shown with red symbols, and lines and non-neutralizing mAbs shown with solid black symbols and lines for IgM and gray symbols and dashed lines for IgG. IgM and IgG control antibodies were used to determine the background of the assay and for which ODs above this line were considered positive. Data are representative from three independent experiments.

    Techniques Used: Isolation, Mouse Assay, Generated, Neutralization, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P
    Figure Legend Snippet: Elevated IgM anti-histone H2A titers correlate with tier 2 HIV-1 neutralization by pristane treated wild-type C57BL/6 mice. Total serum concentrations of (A) IgM, (B) IgG, and (C) relative titers of serum IgM anti-H2A are shown for naive (open circles) B6 mice or B6 mice treated 30 d with pristane only (gray circles) and subsequently immunized 2X or 3X (black circles) with alum alone or Env + alum. Serum from individual B6 mice (regardless of treatment with pristane alone, alum alone, or Env + alum) were separated based on neutralization of ≥1 tier 2 HIV-1 strains and measured for (D) IgM anti-H2A or (E) IgM anti-H2B relative titers. Mice neutralizing only tier 1 strains were included in the tier 2 nonneutralizer group (mostly 3X). Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. All P-values were calculated using Student’s t test assuming unequal variances. *, P

    Techniques Used: Neutralization, Mouse Assay

    B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P
    Figure Legend Snippet: B6.Sle123 HIV-1 neutralizers harbor elevated levels of IgM anti-histone H2A. (A) Sera from B6 (open), B6.Sle123 nonneutralizers (gray), and neutralizers (black) were interrogated with an autoantigen array and results for the IgM reactive with the indicated anti-DNA antigens (top left), RNA-binding proteins (bottom left), and anti-histone antigens (top right), as well as IgG anti-histone antigens (bottom right) are shown. Relative serum titers of (B) IgM anti-H2A (left) and anti-H2B (right) measured by ELISA in B6.Sle123 neutralizers and nonneutralizers. Each symbol represents measurements for one mouse, and all data are plotted as the arithmetic mean ± SEM. P-values were calculated using the Mann-Whitney nonparametric test; *, P

    Techniques Used: RNA Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    6) Product Images from "The Bis-Electrophile Diepoxybutane Cross-links DNA to Human Histones but Does Not Result in Enhanced Mutagenesis in Recombinant Systems"

    Article Title: The Bis-Electrophile Diepoxybutane Cross-links DNA to Human Histones but Does Not Result in Enhanced Mutagenesis in Recombinant Systems

    Journal: Chemical research in toxicology

    doi: 10.1021/tx900037u

    DNA binding assays. Samples containing purified histone H2b or AGT were incubated with 32 P-5’-end-labeled 16-mer at 23 °C for 30 min. A 15% (w/v) native polyacrylamide gel electrophoresis system was used to separate protein-DNA complexes
    Figure Legend Snippet: DNA binding assays. Samples containing purified histone H2b or AGT were incubated with 32 P-5’-end-labeled 16-mer at 23 °C for 30 min. A 15% (w/v) native polyacrylamide gel electrophoresis system was used to separate protein-DNA complexes

    Techniques Used: Binding Assay, Purification, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

    Cross-linking of purified histone H2b to oligonucleotides by diepoxybutane. (A) Gel shift assays were performed by incubating histone H2b (1 μg) and 32 P-5’-endlabeled 16-mer oligonucleotide in reactions containing DMSO (
    Figure Legend Snippet: Cross-linking of purified histone H2b to oligonucleotides by diepoxybutane. (A) Gel shift assays were performed by incubating histone H2b (1 μg) and 32 P-5’-endlabeled 16-mer oligonucleotide in reactions containing DMSO (

    Techniques Used: Purification, Electrophoretic Mobility Shift Assay

    MS analysis of reactions between bis -electrophiles and histones H2b and H3. (A) Purified histone H2b (1 μg) or (B) histone H3 (1 μg) was incubated with 10 nM to 10 mM 1,2-dibromoethane or diepoxybutane for 1 h at 37 °C and digested
    Figure Legend Snippet: MS analysis of reactions between bis -electrophiles and histones H2b and H3. (A) Purified histone H2b (1 μg) or (B) histone H3 (1 μg) was incubated with 10 nM to 10 mM 1,2-dibromoethane or diepoxybutane for 1 h at 37 °C and digested

    Techniques Used: Mass Spectrometry, Purification, Incubation

    Histone H2b expression in E. coli does not enhance mutagenesis by bis -electrophiles. (A) Recombinant expression of histone H2b in TRG8 cells was quantified at various time points after induction by immunoblotting. Expression of histone H2b (100 nM) was
    Figure Legend Snippet: Histone H2b expression in E. coli does not enhance mutagenesis by bis -electrophiles. (A) Recombinant expression of histone H2b in TRG8 cells was quantified at various time points after induction by immunoblotting. Expression of histone H2b (100 nM) was

    Techniques Used: Expressing, Mutagenesis, Recombinant

    Detection of in vivo DNA-histone H2b cross-links. E. coli TRG8 cells expressing histone H2b or containing an empty pINIII vector were treated with 0, 0.032, or 0.2 mM diepoxybutane for 90 min at 37 °C. The genomic DNA from 1 mL cultures was isolated,
    Figure Legend Snippet: Detection of in vivo DNA-histone H2b cross-links. E. coli TRG8 cells expressing histone H2b or containing an empty pINIII vector were treated with 0, 0.032, or 0.2 mM diepoxybutane for 90 min at 37 °C. The genomic DNA from 1 mL cultures was isolated,

    Techniques Used: In Vivo, Expressing, Plasmid Preparation, Isolation

    Proposed products of reactions between lysine, diepoxybutane, and guanine. A targeted MS search for protein adducts was performed on the tryptic peptides from samples of purified histone H2b incubated with diepoxybutane. Reactions containing histone H2b,
    Figure Legend Snippet: Proposed products of reactions between lysine, diepoxybutane, and guanine. A targeted MS search for protein adducts was performed on the tryptic peptides from samples of purified histone H2b incubated with diepoxybutane. Reactions containing histone H2b,

    Techniques Used: Mass Spectrometry, Purification, Incubation

    7) Product Images from "Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos"

    Article Title: Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

    Journal: eLife

    doi: 10.7554/eLife.23326

    Onset of transcription coincides with a reduction in nuclear histone concentration. ( A ) Changes in total animal cap volume, the fraction of the animal cap volume occupied by nuclei, and the size of individual nuclei for indicated stages. Volumes were measured by lightsheet microscopy of embryos injected with mRNA encoding H4-sfGFP and subsequent automated image analysis (error bars represent SEM, n = 3 embryos; offspring of transgenic PCNA-RFP was used to monitor integrity of imaged nuclei via a second color channel). ( B ) Image sequences showing nuclear import of H4-sfGFP fusion protein at 32- to 64- and 128-cell stages. Color scaling was kept constant and linear within each stage. Images show a representative maximum z-projection of a subset of a 3D microscopy stack of one of the embryos used in A. ( C ) The linear range of Western blots was determined using chromatin of sphere-stage embryos. Plotted are observed versus expected fold differences in H2B intensity using different numbers of embryos (n ≥ 3). Band intensities of test stages ( Figure 4F ) were only used for the analysis when they fell within the linear range. DOI: http://dx.doi.org/10.7554/eLife.23326.015
    Figure Legend Snippet: Onset of transcription coincides with a reduction in nuclear histone concentration. ( A ) Changes in total animal cap volume, the fraction of the animal cap volume occupied by nuclei, and the size of individual nuclei for indicated stages. Volumes were measured by lightsheet microscopy of embryos injected with mRNA encoding H4-sfGFP and subsequent automated image analysis (error bars represent SEM, n = 3 embryos; offspring of transgenic PCNA-RFP was used to monitor integrity of imaged nuclei via a second color channel). ( B ) Image sequences showing nuclear import of H4-sfGFP fusion protein at 32- to 64- and 128-cell stages. Color scaling was kept constant and linear within each stage. Images show a representative maximum z-projection of a subset of a 3D microscopy stack of one of the embryos used in A. ( C ) The linear range of Western blots was determined using chromatin of sphere-stage embryos. Plotted are observed versus expected fold differences in H2B intensity using different numbers of embryos (n ≥ 3). Band intensities of test stages ( Figure 4F ) were only used for the analysis when they fell within the linear range. DOI: http://dx.doi.org/10.7554/eLife.23326.015

    Techniques Used: Concentration Assay, Microscopy, Injection, Transgenic Assay, Western Blot

    8) Product Images from "Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease"

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease

    Journal: American Journal of Respiratory and Critical Care Medicine

    doi: 10.1164/rccm.201302-0342OC

    Comparison of gene expression levels of H3.3, H3.1, and H2B in the individual lung tissue from subjects with chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease [GOLD] II and IV) and control subjects (ex-smokers), as determined by the TaqMan quantitative polymerase chain reaction assay system, to measure mRNA. Gene expression levels were not different when GOLD II was compared with control subjects. Only twofold increase in H3.3 genes expression (H3F3A and H3F3B) was observed when GOLD IV was compared with the control subjects. P
    Figure Legend Snippet: Comparison of gene expression levels of H3.3, H3.1, and H2B in the individual lung tissue from subjects with chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease [GOLD] II and IV) and control subjects (ex-smokers), as determined by the TaqMan quantitative polymerase chain reaction assay system, to measure mRNA. Gene expression levels were not different when GOLD II was compared with control subjects. Only twofold increase in H3.3 genes expression (H3F3A and H3F3B) was observed when GOLD IV was compared with the control subjects. P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    ( A ) Comparison of the cytotoxicity of enriched H3.3 from chronic obstructive pulmonary disease (COPD) nuclear lung lysates and recombinant H3.3. The primary lung cell damage was measured by a fluorescent detection of annexin V binding, 24 hours after incubation, with H3.3 extracted from the COPD lungs and recombinant H3.3 (10 μg/ml). ( B ) The primary cells were treated with increasing concentrations of recombinant H3.3 or ( C ) human recombinant histones H2B, H3.3, and H4 (10 μg/ml) for 24 hours. Cytotoxicity was evaluated by measuring bound fluorescent annexin V using a fluorescent reader and was normalized against the control cells without histone H3.3. ( D ) Comparison of the cell death in primary lung cells endogenously overexpressing recombinant H3.3 and the cells transfected to secrete recombinant H3.3. Cells transfected with empty plasmid were used as a control. P
    Figure Legend Snippet: ( A ) Comparison of the cytotoxicity of enriched H3.3 from chronic obstructive pulmonary disease (COPD) nuclear lung lysates and recombinant H3.3. The primary lung cell damage was measured by a fluorescent detection of annexin V binding, 24 hours after incubation, with H3.3 extracted from the COPD lungs and recombinant H3.3 (10 μg/ml). ( B ) The primary cells were treated with increasing concentrations of recombinant H3.3 or ( C ) human recombinant histones H2B, H3.3, and H4 (10 μg/ml) for 24 hours. Cytotoxicity was evaluated by measuring bound fluorescent annexin V using a fluorescent reader and was normalized against the control cells without histone H3.3. ( D ) Comparison of the cell death in primary lung cells endogenously overexpressing recombinant H3.3 and the cells transfected to secrete recombinant H3.3. Cells transfected with empty plasmid were used as a control. P

    Techniques Used: Recombinant, Binding Assay, Incubation, Transfection, Plasmid Preparation

    9) Product Images from "Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects"

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05451-7

    Histone homocysteinylation is a common modification among different tissues and species. a A typical HPLC-MS/MS spectra of a tryptic peptide ‘RGGVK Hcy RISGLIYEETR’ harboring H4K44 homocystylation, derived from human brain. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. b Schematic illustration of homocysteinylation sites of histone lysine residues in human normal brain samples identified using HPLC-MS/MS. The red diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. c Verification of anti-KHcy antibody. The homocysteinylation levels of BSA (Bovine Serum Album) and KHcy modified BSA were detected with anti-KHcy antibody under the presence of 0, 2, or 5 µg/ml of Hcy modified lysine (KHcy as competitor). CBB Coomassie Brilliant Blue staining. These test repeated for 3 times and the quantitation of the western blotting showed on right. In the BSA group, the relative K-Hcy levels were 1 ± 0.05, 1.15 ± 0.10; 1.11 ± 0.78. In the BSA-Hcy group, the relative K-Hcy levels were 10.88 ± 1.02, 5.48 ± 0.34; 1.39 ± 0.21. d Verification of specificity of the anti-K-Hcy antibody. Western blotting assay was carried out by incubating the anti-KHcy antibody with unmodified OVA (ovalbumin), acetylated-OVA, succinylated-OVA, or K-Hcy-OVA. e Western blotting analysis for the detection of H3 homocystylations in samples from a variety of human fetal tissues, including brain, spinal cord, heart, liver, lung, kidney, muscle, and skin. Anti-Hcy: rabbit polyclonal anti-Hcy antibody; Anti-H3: rabbit polyclonal anti-H3 antibody. f Presence of H3 homocysteinylation in different species, including D. melanogaster , Zebra fish, Gallus gallus brain, mouse brain, and human fetal brain, demonstrated using western blotting with rabbit polyclonal anti-Hcy and anti-H3 antibodies
    Figure Legend Snippet: Histone homocysteinylation is a common modification among different tissues and species. a A typical HPLC-MS/MS spectra of a tryptic peptide ‘RGGVK Hcy RISGLIYEETR’ harboring H4K44 homocystylation, derived from human brain. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. b Schematic illustration of homocysteinylation sites of histone lysine residues in human normal brain samples identified using HPLC-MS/MS. The red diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. c Verification of anti-KHcy antibody. The homocysteinylation levels of BSA (Bovine Serum Album) and KHcy modified BSA were detected with anti-KHcy antibody under the presence of 0, 2, or 5 µg/ml of Hcy modified lysine (KHcy as competitor). CBB Coomassie Brilliant Blue staining. These test repeated for 3 times and the quantitation of the western blotting showed on right. In the BSA group, the relative K-Hcy levels were 1 ± 0.05, 1.15 ± 0.10; 1.11 ± 0.78. In the BSA-Hcy group, the relative K-Hcy levels were 10.88 ± 1.02, 5.48 ± 0.34; 1.39 ± 0.21. d Verification of specificity of the anti-K-Hcy antibody. Western blotting assay was carried out by incubating the anti-KHcy antibody with unmodified OVA (ovalbumin), acetylated-OVA, succinylated-OVA, or K-Hcy-OVA. e Western blotting analysis for the detection of H3 homocystylations in samples from a variety of human fetal tissues, including brain, spinal cord, heart, liver, lung, kidney, muscle, and skin. Anti-Hcy: rabbit polyclonal anti-Hcy antibody; Anti-H3: rabbit polyclonal anti-H3 antibody. f Presence of H3 homocysteinylation in different species, including D. melanogaster , Zebra fish, Gallus gallus brain, mouse brain, and human fetal brain, demonstrated using western blotting with rabbit polyclonal anti-Hcy and anti-H3 antibodies

    Techniques Used: Modification, High Performance Liquid Chromatography, Mass Spectrometry, Derivative Assay, Sequencing, Staining, Quantitation Assay, Western Blot, Fluorescence In Situ Hybridization

    Direct in vitro histone homocysteinylation by HTL. a Schematic illustration of protein modification by HTL. b Dot-blot analysis of histone homocysteinylation by HTL. The unmodified histones H3, H4, H2a, and H2b expressed from E. coli were used. Top panel: four histones were incubated with 5 mM HTL for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. ( + : positive control, tubulin antibody diluted 1:1000 was used as the positive control; – : negative control, sodium phosphate buffer was used as the negative control); Middle panel: histone H3 was treated with 5 mM HTL for 2, 6, and 14 h, respectively, and histone homocysteinylation was detected using anti-Hcy antibodies. Bottom panel: histone H3 was treated with 0.5 mM, 1 mM, 5 mM, and 10 mM HTL respectively for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. c MALDI analysis of unmodified H3 from E. coli with (bottom) or without (top) in vitro HTL treatment. The undigested H3 display a major peak of about 15KD. Additional major peaks greater than 15KD are seen in HTL-treated H3 samples. The difference in molecular mass between the adjacent two peaks is in the proximity of 3 Hcy modifications, indicating that multiple, simultaneous KHcy modifications may exist on H3 during HTL treatment. The x and y axes represent m / z and relative ion intensity, respectively. d A typical HPLC-MS/MS spectra of a tryptic peptide ‘GVLK Hcy VFLENVIR’ derived from HTL-treated H4 with homocystylation at H4K59 site. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. e Illustration of histone homocysteinylation sites identified by HPLC-MS/MS analysis on unmodified core histones treated with HTL. The green diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. Homocysteinylation sites, present both naturally in normal human brain samples (Fig. 1b ) and after in vitro HTL treatment are marked with a red dot
    Figure Legend Snippet: Direct in vitro histone homocysteinylation by HTL. a Schematic illustration of protein modification by HTL. b Dot-blot analysis of histone homocysteinylation by HTL. The unmodified histones H3, H4, H2a, and H2b expressed from E. coli were used. Top panel: four histones were incubated with 5 mM HTL for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. ( + : positive control, tubulin antibody diluted 1:1000 was used as the positive control; – : negative control, sodium phosphate buffer was used as the negative control); Middle panel: histone H3 was treated with 5 mM HTL for 2, 6, and 14 h, respectively, and histone homocysteinylation was detected using anti-Hcy antibodies. Bottom panel: histone H3 was treated with 0.5 mM, 1 mM, 5 mM, and 10 mM HTL respectively for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. c MALDI analysis of unmodified H3 from E. coli with (bottom) or without (top) in vitro HTL treatment. The undigested H3 display a major peak of about 15KD. Additional major peaks greater than 15KD are seen in HTL-treated H3 samples. The difference in molecular mass between the adjacent two peaks is in the proximity of 3 Hcy modifications, indicating that multiple, simultaneous KHcy modifications may exist on H3 during HTL treatment. The x and y axes represent m / z and relative ion intensity, respectively. d A typical HPLC-MS/MS spectra of a tryptic peptide ‘GVLK Hcy VFLENVIR’ derived from HTL-treated H4 with homocystylation at H4K59 site. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. e Illustration of histone homocysteinylation sites identified by HPLC-MS/MS analysis on unmodified core histones treated with HTL. The green diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. Homocysteinylation sites, present both naturally in normal human brain samples (Fig. 1b ) and after in vitro HTL treatment are marked with a red dot

    Techniques Used: In Vitro, Modification, Dot Blot, Incubation, Positive Control, Negative Control, High Performance Liquid Chromatography, Mass Spectrometry, Derivative Assay, Sequencing

    10) Product Images from "Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿"

    Article Title: Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00290-10

    (A) The histone H2B R102A mutant represses the expression of telomere-proximal genes. Depicted is the chromosome plot of the genome-wide expression changes for the histone H2B R102A mutant relative to the wild type. A histogram of the fraction of genes whose mRNA levels are up- or downregulated is plotted as a function of their distance from a chromosome end. (B) The histone H2B R102A mutation causes spreading of Sir4 beyond the telomeres. ChIP analysis was used to examine the level of Sir4 protein binding to five regions on telomere V-L in wild-type and histone H2B R102A and K111A mutant strains. An untagged strain was used as a control, and Sir4 binding ratios were normalized to ACT1 ) and examined by Western blotting. The level of histone H2B protein was not significantly changed in the histone mutant strains compared to that in the wild type. Histone H3 was used as a loading control. α, anti.
    Figure Legend Snippet: (A) The histone H2B R102A mutant represses the expression of telomere-proximal genes. Depicted is the chromosome plot of the genome-wide expression changes for the histone H2B R102A mutant relative to the wild type. A histogram of the fraction of genes whose mRNA levels are up- or downregulated is plotted as a function of their distance from a chromosome end. (B) The histone H2B R102A mutation causes spreading of Sir4 beyond the telomeres. ChIP analysis was used to examine the level of Sir4 protein binding to five regions on telomere V-L in wild-type and histone H2B R102A and K111A mutant strains. An untagged strain was used as a control, and Sir4 binding ratios were normalized to ACT1 ) and examined by Western blotting. The level of histone H2B protein was not significantly changed in the histone mutant strains compared to that in the wild type. Histone H3 was used as a loading control. α, anti.

    Techniques Used: Mutagenesis, Expressing, Genome Wide, Chromatin Immunoprecipitation, Protein Binding, Binding Assay, Western Blot

    Mutations in histone H2B core residues render yeast cells sensitive to DNA-damaging agents. (A) Growth phenotypes of histone H2B alanine mutant strains in the presence of the DNA-damaging agent MMS. Tenfold serial dilutions of each strain (starting with approximately 5 × 10 5 cells) were spotted on SC-Trp medium or SC-Trp medium containing the indicated concentrations of MMS. (B) Histone H2B core mutant strains were examined for sensitivity to UV irradiation at doses of 0, 50, and 100 J/m 2 . The percentage of viable cells in each strain was plotted as a function of UV dose. Only the histone H2B alanine mutant strains with a significant UV sensitivity phenotype are depicted. WT, wild type.
    Figure Legend Snippet: Mutations in histone H2B core residues render yeast cells sensitive to DNA-damaging agents. (A) Growth phenotypes of histone H2B alanine mutant strains in the presence of the DNA-damaging agent MMS. Tenfold serial dilutions of each strain (starting with approximately 5 × 10 5 cells) were spotted on SC-Trp medium or SC-Trp medium containing the indicated concentrations of MMS. (B) Histone H2B core mutant strains were examined for sensitivity to UV irradiation at doses of 0, 50, and 100 J/m 2 . The percentage of viable cells in each strain was plotted as a function of UV dose. Only the histone H2B alanine mutant strains with a significant UV sensitivity phenotype are depicted. WT, wild type.

    Techniques Used: Mutagenesis, Irradiation

    ). DNA is in magenta, histone residues are in cyan, and Sir3 protein is in green. The LRS domain is highlighted in yellow, histone H2B R102 is depicted in dark blue, histone H2B K111 is shown in red, and Sir3 R210 is highlighted in orange. Panels D to F are magnified or rotated images of the Sir3-H2B interactions depicted in panel C, with the DNA hidden from view. (G) Sequence alignment of the BAH domain protein sequences of Sir3 and Orc1 from Saccharomyces cerevisiae , Saccharomyces paradoxus , Saccharomyces bayanus , and Saccharomyces castellii ). The Sir3 R210 residue is highlighted in orange and labeled with an orange asterisk.
    Figure Legend Snippet: ). DNA is in magenta, histone residues are in cyan, and Sir3 protein is in green. The LRS domain is highlighted in yellow, histone H2B R102 is depicted in dark blue, histone H2B K111 is shown in red, and Sir3 R210 is highlighted in orange. Panels D to F are magnified or rotated images of the Sir3-H2B interactions depicted in panel C, with the DNA hidden from view. (G) Sequence alignment of the BAH domain protein sequences of Sir3 and Orc1 from Saccharomyces cerevisiae , Saccharomyces paradoxus , Saccharomyces bayanus , and Saccharomyces castellii ). The Sir3 R210 residue is highlighted in orange and labeled with an orange asterisk.

    Techniques Used: Sequencing, Labeling

    Effects of histone H2B core mutants on telomeric and HML silencing. (A) Tenfold serial dilutions (starting with approximately 5 × 10 5 cells) of each histone H2B alanine mutant strain were spotted on SC-Trp, Sc-Trp-Ura, or SC-Trp+5-FOA agar plates, as indicated. (B) Quantification of telomeric silencing phenotypes in the histone H2B mutant strains. Each data point represents the average of three replicate experiments. Error bars indicate the standard deviations, and strains that exhibit sensitivity significantly ( P
    Figure Legend Snippet: Effects of histone H2B core mutants on telomeric and HML silencing. (A) Tenfold serial dilutions (starting with approximately 5 × 10 5 cells) of each histone H2B alanine mutant strain were spotted on SC-Trp, Sc-Trp-Ura, or SC-Trp+5-FOA agar plates, as indicated. (B) Quantification of telomeric silencing phenotypes in the histone H2B mutant strains. Each data point represents the average of three replicate experiments. Error bars indicate the standard deviations, and strains that exhibit sensitivity significantly ( P

    Techniques Used: Mutagenesis

    Western blot analysis of potential histone H2B posttranslational modification sites. (A) Custom antibodies were designed to detect either dimethylation or acetylation of yeast histone H2B K111. Peptide dot blots confirm that each antibody has a higher specificity for the modified peptide than for the unmodified peptide and that the anti-H2B K111ac antibody does not nonspecifically bind to a histone H3 peptide dimethylated at lysine 4. (B) Custom anti-H2B K111me2 and anti-H2B K111Ac antibodies do not specifically detect modifications in yeast histone H2B. Wild-type and histone H2B K111 mutant yeast strain whole-cell extracts were examined by Western blotting using anti-H2B K111me2, anti-H2B K111Ac, or anti-H2B antibodies. (C) Histones were purified from wild-type yeast cells and analyzed by Western blotting using the H2B K111me2 antibody. (D) Human histone H2B K46 is acetylated and dimethylated. Anti-H2B K46Ac and anti-H2B K46me2 antibodies were used to confirm the presence of both modifications on human histone H2B (hH2B) K46 in MCF7 breast cancer cell extracts. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) or valproic acid (VPA) resulted in an increase in histone H2B K46 acetylation. Neither acetylation nor dimethylation was detected on recombinant histone H2B (rH2B).
    Figure Legend Snippet: Western blot analysis of potential histone H2B posttranslational modification sites. (A) Custom antibodies were designed to detect either dimethylation or acetylation of yeast histone H2B K111. Peptide dot blots confirm that each antibody has a higher specificity for the modified peptide than for the unmodified peptide and that the anti-H2B K111ac antibody does not nonspecifically bind to a histone H3 peptide dimethylated at lysine 4. (B) Custom anti-H2B K111me2 and anti-H2B K111Ac antibodies do not specifically detect modifications in yeast histone H2B. Wild-type and histone H2B K111 mutant yeast strain whole-cell extracts were examined by Western blotting using anti-H2B K111me2, anti-H2B K111Ac, or anti-H2B antibodies. (C) Histones were purified from wild-type yeast cells and analyzed by Western blotting using the H2B K111me2 antibody. (D) Human histone H2B K46 is acetylated and dimethylated. Anti-H2B K46Ac and anti-H2B K46me2 antibodies were used to confirm the presence of both modifications on human histone H2B (hH2B) K46 in MCF7 breast cancer cell extracts. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) or valproic acid (VPA) resulted in an increase in histone H2B K46 acetylation. Neither acetylation nor dimethylation was detected on recombinant histone H2B (rH2B).

    Techniques Used: Western Blot, Modification, Mutagenesis, Purification, Histone Deacetylase Assay, Recombinant

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    Article Title: Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos
    Article Snippet: .. Histone cocktail Recombinant histones were of human origin and produced in E. Coli (NEB, Ipswich, MA; 1 mg/ml: H3.1 M2503S, H4 M2504S, H2A M2502S, H2B M2505S). ..

    Concentration Assay:

    Article Title: Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region
    Article Snippet: .. Recombinant histone H2B (New England Biolabs) from human was used as a MST1 substrate at a concentration of 1 mg/mL. .. Radiolabeled [γ-32 P]ATP (3000 Ci/mmol) was purchased from Perkin-Elmer Life Sciences.

    Recombinant:

    Article Title: APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells
    Article Snippet: .. Recombinant human histone H2B was from New England Biolabs. .. A549 cell lines stably transfected with pSUPER-APLF, designated A549-APLFKD , were maintained in minimal essential medium supplemented with 15% fetal calf serum, 1.5 mg/ml G418, 2 mM l -glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin.

    Article Title: Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos
    Article Snippet: .. Histone cocktail Recombinant histones were of human origin and produced in E. Coli (NEB, Ipswich, MA; 1 mg/ml: H3.1 M2503S, H4 M2504S, H2A M2502S, H2B M2505S). ..

    Article Title: Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region
    Article Snippet: .. Recombinant histone H2B (New England Biolabs) from human was used as a MST1 substrate at a concentration of 1 mg/mL. .. Radiolabeled [γ-32 P]ATP (3000 Ci/mmol) was purchased from Perkin-Elmer Life Sciences.

    Article Title: Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming \u03c9-NG-Monomethylated Arginine Residues
    Article Snippet: .. Histones H2A (M2502S), H2B (M2505S), H3.2 (M2506S), and H4 (M2504S) are human recombinant species from New England BioLabs, and myelin basic protein from bovine brain was obtained from Sigma (M1891). .. Purified Myc-PRMT5 from HEK293 cells was a kind gift from Dr. Jill Butler (University of Texas MD Anderson Cancer Center, Houston, TX).

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease
    Article Snippet: .. We used the primary human bronchial epithelial cells to evaluate in vitro the cytotoxicity effect of H3.3 purified from subjects with COPD and human recombinant histones H2B, H3.3, and H4 (New England Biolabs, Ipswich, MA). ..

    Article Title: Breaching peripheral tolerance promotes the production of HIV-1–neutralizing antibodies
    Article Snippet: .. For Histone H2A- and H2B-reactive IgM ELISAs, plates were coated with 1 µg/ml of human recombinant histone H2A or H2B (New England Biolabs) in PBS pH 7.4. ..

    Article Title: Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia
    Article Snippet: .. Histone application Human recombinant histones H1, H2A, H2B, H3 and H4 were obtained from New England Biolabs. .. Additionally, histone H1 purified from calf thymus, andXenopus laevis recombinant histones H2A, H2B, H3 and H4 were obtained from Millipore.

    In Vitro:

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease
    Article Snippet: .. We used the primary human bronchial epithelial cells to evaluate in vitro the cytotoxicity effect of H3.3 purified from subjects with COPD and human recombinant histones H2B, H3.3, and H4 (New England Biolabs, Ipswich, MA). ..

    Purification:

    Article Title: Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease
    Article Snippet: .. We used the primary human bronchial epithelial cells to evaluate in vitro the cytotoxicity effect of H3.3 purified from subjects with COPD and human recombinant histones H2B, H3.3, and H4 (New England Biolabs, Ipswich, MA). ..

    Article Title: The Bis-Electrophile Diepoxybutane Cross-links DNA to Human Histones but Does Not Result in Enhanced Mutagenesis in Recombinant Systems
    Article Snippet: .. Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA). .. Bovine DNase I and single-stranded calf thymus DNA-cellulose was purchased from Sigma-Aldrich (St. Louis, MO).

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    New England Biolabs histone h2b human recombinant
    GST-PRMT7 can methylate histone <t>H2B</t> producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μ m ) with histone H2B (10 μg; 12.1 μ m ) was carried out for 1 (■), 5 (♦), and 25 h (●)
    Histone H2b Human Recombinant, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GST-PRMT7 can methylate histone H2B producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μ m ) with histone H2B (10 μg; 12.1 μ m ) was carried out for 1 (■), 5 (♦), and 25 h (●)

    Journal: The Journal of Biological Chemistry

    Article Title: Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming \u03c9-NG-Monomethylated Arginine Residues

    doi: 10.1074/jbc.M111.336271

    Figure Lengend Snippet: GST-PRMT7 can methylate histone H2B producing only ω-MMA. In vitro methylation reaction of GST-PRMT7 (15 μg; 2.4 μ m ) with histone H2B (10 μg; 12.1 μ m ) was carried out for 1 (■), 5 (♦), and 25 h (●)

    Article Snippet: Histones H2A (M2502S), H2B (M2505S), H3.2 (M2506S), and H4 (M2504S) are human recombinant species from New England BioLabs, and myelin basic protein from bovine brain was obtained from Sigma (M1891).

    Techniques: In Vitro, Methylation

    The tandem zinc finger domain of APLF binds poly(ADP-ribosyl)ated proteins in vitro. (A) Recombinant human His-APLF proteins. One microgram of recombinant His-APLF and the indicated mutated derivatives were fractionated by SDS-PAGE and stained with Coomassie blue. A schematic of the recombinant His-APLF proteins is shown (bottom). “H-” and “FHA” denote the decahistidine tag and FHA domain, respectively. The C-terminal acidic tail (gray box) and tandem ZNF domains (black boxes: *, ZNF1; **, ZNF2) are also indicated. Dotted boxes denote a mutated ZNF. Amino acid positions are indicated at the top. (B) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated (pADPr-h1) or not (h1) as described above and incubated in the absence (−) or presence of 17.5 nM of the indicated His-APLF protein for 30 min on ice. Bound His-APLF was quantified as described above. (C) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated and incubated with the indicated concentration of the indicated His-APLF protein for 30 min at 37°C. Bound His-APLF was quantified as described above. (D) Two micrograms of BSA, calf thymus histone H1, recombinant human histone H2B, or recombinant human PARP-1 was slot blotted onto nitrocellulose and mock treated (−pADPr) or poly(ADP-ribosyl)ated (+pADPr) with recombinant human PARP-1 (P1). Filters were incubated with 35 nM His-APLF, and after extensive washing, filter-bound APLF was detected with anti-His-Tag MAb and appropriate secondary antibody. Error bars show standard errors of the mean.

    Journal: Molecular and Cellular Biology

    Article Title: APLF (C2orf13) Is a Novel Component of Poly(ADP-Ribose) Signaling in Mammalian Cells

    doi: 10.1128/MCB.02243-07

    Figure Lengend Snippet: The tandem zinc finger domain of APLF binds poly(ADP-ribosyl)ated proteins in vitro. (A) Recombinant human His-APLF proteins. One microgram of recombinant His-APLF and the indicated mutated derivatives were fractionated by SDS-PAGE and stained with Coomassie blue. A schematic of the recombinant His-APLF proteins is shown (bottom). “H-” and “FHA” denote the decahistidine tag and FHA domain, respectively. The C-terminal acidic tail (gray box) and tandem ZNF domains (black boxes: *, ZNF1; **, ZNF2) are also indicated. Dotted boxes denote a mutated ZNF. Amino acid positions are indicated at the top. (B) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated (pADPr-h1) or not (h1) as described above and incubated in the absence (−) or presence of 17.5 nM of the indicated His-APLF protein for 30 min on ice. Bound His-APLF was quantified as described above. (C) Multiwell dishes coated with calf thymus histone H1 were poly(ADP-ribosyl)ated and incubated with the indicated concentration of the indicated His-APLF protein for 30 min at 37°C. Bound His-APLF was quantified as described above. (D) Two micrograms of BSA, calf thymus histone H1, recombinant human histone H2B, or recombinant human PARP-1 was slot blotted onto nitrocellulose and mock treated (−pADPr) or poly(ADP-ribosyl)ated (+pADPr) with recombinant human PARP-1 (P1). Filters were incubated with 35 nM His-APLF, and after extensive washing, filter-bound APLF was detected with anti-His-Tag MAb and appropriate secondary antibody. Error bars show standard errors of the mean.

    Article Snippet: Recombinant human histone H2B was from New England Biolabs.

    Techniques: In Vitro, Recombinant, SDS Page, Staining, Incubation, Concentration Assay

    Kinetic analysis of MST1 protein constructs. (A) Lineweaver–Burk plot comparing the activity of MST1-FL and the kinase domain (MST1-KD) toward FoxO1. (B) Lineweaver–Burk plot depicting the activity of MST-KD toward histone H2B. (C) Lineweaver–Burk plot depicting the activity of MST1-FL toward histone H2B. (D) Summary of the various steady state parameters for the plots shown in panels A–C. (E and F) Autoradiography gel showing the activity of MST1-FL and MST1-KD toward FoxO1 and histone H2B, respectively. The gel shows the incorporation of the γ- 32 P label as a function of substrate concentration. All reactions were carried out for 15 min and then stopped with the addition of SDS–PAGE loading buffer.

    Journal: Biochemistry

    Article Title: Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region

    doi: 10.1021/bi800309m

    Figure Lengend Snippet: Kinetic analysis of MST1 protein constructs. (A) Lineweaver–Burk plot comparing the activity of MST1-FL and the kinase domain (MST1-KD) toward FoxO1. (B) Lineweaver–Burk plot depicting the activity of MST-KD toward histone H2B. (C) Lineweaver–Burk plot depicting the activity of MST1-FL toward histone H2B. (D) Summary of the various steady state parameters for the plots shown in panels A–C. (E and F) Autoradiography gel showing the activity of MST1-FL and MST1-KD toward FoxO1 and histone H2B, respectively. The gel shows the incorporation of the γ- 32 P label as a function of substrate concentration. All reactions were carried out for 15 min and then stopped with the addition of SDS–PAGE loading buffer.

    Article Snippet: Recombinant histone H2B (New England Biolabs) from human was used as a MST1 substrate at a concentration of 1 mg/mL.

    Techniques: Construct, Activity Assay, Microscale Thermophoresis, Autoradiography, Concentration Assay, SDS Page

    MST1 preparation and characterization. (A) The construct designs for the various protein fragments of MST1 used for this study are depicted. The various domains are abbreviated as follows: KD, kinase domain; RR, regulatory region; and DD, dimerization domain. (B) SDS page gel of the various recombinant protein constructs used in this study. The abbreviations are as follows: MST1-FL, full-length MST1; MST1-KD/RR, MST1 kinase with the regulatory region, spanning residues 1–380; and MST1-FL (TM), MST1-FL triple mutant with residues S340, T346, and T348 mutated to alanine. (C) Autoradiography gel showing the activity of MST1 toward phosphorylation of its substrates, FoxO1 and histone H2B. The signal depicted is the incorporation of the γ- 32 P label onto FoxO and histone H2B. The autophosphorylation signal of MST1 is also visible as indicated. (D) Autoradiograph showing the effect of DNA binding on the phosphorylation activity of FoxO1 by MST1-FL in the presence and absence of its cognate DNA, the Daf-16 binding element

    Journal: Biochemistry

    Article Title: Biochemical Analysis of MST1 Kinase: Elucidation of a C-Terminal Regulatory Region

    doi: 10.1021/bi800309m

    Figure Lengend Snippet: MST1 preparation and characterization. (A) The construct designs for the various protein fragments of MST1 used for this study are depicted. The various domains are abbreviated as follows: KD, kinase domain; RR, regulatory region; and DD, dimerization domain. (B) SDS page gel of the various recombinant protein constructs used in this study. The abbreviations are as follows: MST1-FL, full-length MST1; MST1-KD/RR, MST1 kinase with the regulatory region, spanning residues 1–380; and MST1-FL (TM), MST1-FL triple mutant with residues S340, T346, and T348 mutated to alanine. (C) Autoradiography gel showing the activity of MST1 toward phosphorylation of its substrates, FoxO1 and histone H2B. The signal depicted is the incorporation of the γ- 32 P label onto FoxO and histone H2B. The autophosphorylation signal of MST1 is also visible as indicated. (D) Autoradiograph showing the effect of DNA binding on the phosphorylation activity of FoxO1 by MST1-FL in the presence and absence of its cognate DNA, the Daf-16 binding element

    Article Snippet: Recombinant histone H2B (New England Biolabs) from human was used as a MST1 substrate at a concentration of 1 mg/mL.

    Techniques: Construct, SDS Page, Recombinant, Mutagenesis, Autoradiography, Activity Assay, Binding Assay

    Linker histone H1 is neurotoxic. Dissociated cortical neurons were cultured for 48 hrs and histones were applied for a further 24 hrs. Cell death was determined by luminescence to measure protease release and was expressed as relative luminescence units (RLU). For clarity RLU means are expressed as +/- 2 x standard deviation (SD). Histone H1 caused dose-dependent death of cortical neurons ( a ) while core histones H2A, H2B, H3 and H4 were without effect up to 200 nM ( b–e ). Cell death as measured by luminescence was confirmed directly via phase contrast microscopy of dissociated cortical neurons in culture ( Figure 4 ).

    Journal: F1000Research

    Article Title: Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia

    doi: 10.12688/f1000research.2-148.v1

    Figure Lengend Snippet: Linker histone H1 is neurotoxic. Dissociated cortical neurons were cultured for 48 hrs and histones were applied for a further 24 hrs. Cell death was determined by luminescence to measure protease release and was expressed as relative luminescence units (RLU). For clarity RLU means are expressed as +/- 2 x standard deviation (SD). Histone H1 caused dose-dependent death of cortical neurons ( a ) while core histones H2A, H2B, H3 and H4 were without effect up to 200 nM ( b–e ). Cell death as measured by luminescence was confirmed directly via phase contrast microscopy of dissociated cortical neurons in culture ( Figure 4 ).

    Article Snippet: Histone application Human recombinant histones H1, H2A, H2B, H3 and H4 were obtained from New England Biolabs.

    Techniques: Cell Culture, Standard Deviation, Microscopy

    Release of histones H1 and H2B from ischaemic adult brain. Adult conditioned medium (ACM) was made by incubating ischaemic brain slices in defined culture medium that was subjected to affinity chromatography. Western blots using antibodies specific to histones H1 and H2B show the presence of histone H1 at 32 kDa (lane 1) and histone H2B at 16 kDa in ACM (lane 3). Protein standards are shown at 32 kDa (H1 lane 2) and at 16 kDa for the monomer and 32 kDa for the dimer (H2B lane 4).

    Journal: F1000Research

    Article Title: Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia

    doi: 10.12688/f1000research.2-148.v1

    Figure Lengend Snippet: Release of histones H1 and H2B from ischaemic adult brain. Adult conditioned medium (ACM) was made by incubating ischaemic brain slices in defined culture medium that was subjected to affinity chromatography. Western blots using antibodies specific to histones H1 and H2B show the presence of histone H1 at 32 kDa (lane 1) and histone H2B at 16 kDa in ACM (lane 3). Protein standards are shown at 32 kDa (H1 lane 2) and at 16 kDa for the monomer and 32 kDa for the dimer (H2B lane 4).

    Article Snippet: Histone application Human recombinant histones H1, H2A, H2B, H3 and H4 were obtained from New England Biolabs.

    Techniques: Affinity Chromatography, Western Blot