h2b  (New England Biolabs)


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    Structured Review

    New England Biolabs h2b
    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 <t>H2B,</t> 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.
    H2b, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h2b - by Bioz Stars, 2022-09
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    Images

    1) 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

    2) 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

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    New England Biolabs recombinant human histone h2b
    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 <t>H2B,</t> 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.
    Recombinant Human Histone H2b, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: 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 (

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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 (

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: 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

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: 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

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: 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,

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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,

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: 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,

    Journal: Chemical research in toxicology

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

    doi: 10.1021/tx900037u

    Figure Lengend 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,

    Article Snippet: Purified human histone H2b was purchased from New England Biolabs (Ipswich, MA).

    Techniques: Mass Spectrometry, Purification, Incubation

    Reduced bactericidal activity of Hs2st-deficient neutrophils. Bone marrow neutrophils (A) and LPS-elicited neutrophils (B) were collected from WT or Hs2St-deficient mice. After challenge with GBS, CFU were measured at the indicated time points by serial plating. Data shown are means ± SEM from one of the two independent experiments. (C) Reactive oxygen species production from bone marrow neutrophils after GBS stimulation detected by a luminol-based assay ( n = 2 mice). (D) Bone marrow neutrophils were directly lysed or stimulated with GBS for 30 min. Supernatants were collected to measure the total neutrophil elastase (top) or released neutrophil elastase (bottom) by Western blot analysis. (E) Whole blood from WT and Hs2st-deficient mice was incubated with pHrodo red-labeled GBS (MOI of 100) for 1 h at 37°C (phagocytosis) or 4°C (control). Neutrophils were fixed and subjected to flow cytometry to measure internalized GBS. (F) LPS-elicited neutrophils were stimulated with GBS at an MOI of 10 for 2 h, washed, fixed, and stained with H2A/H2B MAb to visualize NET formation. Differences between groups were calculated by an unpaired t test. **, P

    Journal: Infection and Immunity

    Article Title: Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity

    doi: 10.1128/IAI.00545-15

    Figure Lengend Snippet: Reduced bactericidal activity of Hs2st-deficient neutrophils. Bone marrow neutrophils (A) and LPS-elicited neutrophils (B) were collected from WT or Hs2St-deficient mice. After challenge with GBS, CFU were measured at the indicated time points by serial plating. Data shown are means ± SEM from one of the two independent experiments. (C) Reactive oxygen species production from bone marrow neutrophils after GBS stimulation detected by a luminol-based assay ( n = 2 mice). (D) Bone marrow neutrophils were directly lysed or stimulated with GBS for 30 min. Supernatants were collected to measure the total neutrophil elastase (top) or released neutrophil elastase (bottom) by Western blot analysis. (E) Whole blood from WT and Hs2st-deficient mice was incubated with pHrodo red-labeled GBS (MOI of 100) for 1 h at 37°C (phagocytosis) or 4°C (control). Neutrophils were fixed and subjected to flow cytometry to measure internalized GBS. (F) LPS-elicited neutrophils were stimulated with GBS at an MOI of 10 for 2 h, washed, fixed, and stained with H2A/H2B MAb to visualize NET formation. Differences between groups were calculated by an unpaired t test. **, P

    Article Snippet: Monolayers of cultured primary lung endothelial cells derived from wild-type (WT) and Hs2st f/f Tie2 Cre + mice were detached using Accutase (Millipore) and incubated with human H2B or H3 (5 μg/ml; New England BioLabs) for 1 h in PBS at 4°C.

    Techniques: Activity Assay, Mouse Assay, Western Blot, Incubation, Labeling, Flow Cytometry, Cytometry, Staining

    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