histone  (New England Biolabs)


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    Name:
    Histone H4 Human Recombinant
    Description:
    Histone H4 Human Recombinant 100 ug
    Catalog Number:
    m2504s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
    Buy from Supplier


    Structured Review

    New England Biolabs histone
    Histone H4 Human Recombinant
    Histone H4 Human Recombinant 100 ug
    https://www.bioz.com/result/histone/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Centrifugation:

    Article Title: A screening method for phosphohistidine phosphatase 1 activity
    Article Snippet: Human recombinant histone H4 was from New England Biolabs, UK. .. Before use, this solvent was changed by centrifugation of 50 μL aliquots through a 210 μL DEAE-Sephacel gel equilibrated in 10 mM HCl for 2 min at 900 g according to the manufacturer's instruction.

    Amplification:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Filtration:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The recombinant protein was first purified using an Ni-nitrilotriacetic acid (NTA) column (Qiagen) under native conditions and then by gel filtration chromatography using a Superdex 26/75 column (Amersham). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Positive Control:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.). .. After 2 h of incubation at 37˚C and 5% CO2 , the THP-1 cells were harvested.

    Synthesized:

    Article Title: Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis *
    Article Snippet: Brdt polyclonal antibody was raised in female BALB/c mice using the C-terminal peptide C-NLAREKEQERRRREAMA (position 911–927 amino acids) synthesized at Bio-Concept Laboratories. .. Recombinant histone H4 (human) was procured from New England Biolabs.

    Article Title: A screening method for phosphohistidine phosphatase 1 activity
    Article Snippet: Non-radioactive phosphoramidate was synthesized by applying the method described for [32 P]phosphoramidate by Buckler and Stock ( ). .. Human recombinant histone H4 was from New England Biolabs, UK.

    Mouse Assay:

    Article Title: Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis *
    Article Snippet: Brdt polyclonal antibody was raised in female BALB/c mice using the C-terminal peptide C-NLAREKEQERRRREAMA (position 911–927 amino acids) synthesized at Bio-Concept Laboratories. .. Recombinant histone H4 (human) was procured from New England Biolabs.

    Autoradiography:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. Phosphorylated histone proteins were detected by autoradiography or Western blotting.

    Cytometry:

    Article Title: Histone H4 induces platelet ballooning and microparticle release during trauma hemorrhage
    Article Snippet: Washed platelets (3 × 108 /mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. .. Platelets were then prepared for flow cytometry or imaging flow cytometry as described in the SI Appendix .

    Quantitative RT-PCR:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: It was preliminarily verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that DNA dose-dependently induced TF mRNA expression in the THP-1 cells (tested at 0, 25 and 50 ng/µl; data not shown) and consequently the highest concentration was used for subsequent experiments. .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.).

    Ubiquitin Assay:

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: Paragraph title: In vitro ubiquitylation assay ... Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast.

    Incubation:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.). .. After 2 h of incubation at 37˚C and 5% CO2 , the THP-1 cells were harvested.

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast. .. Reactions were carried out in a total volume of 15µl and incubated for 1h at 30°C, following which 5µl of 4x SDS-PAGE loading buffer was added and the ubiquitylated proteins and the entire reaction was resolved on a 18% SDS-polyacrylamide gel.

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: Recombinant histone H4 was purchased from NEB (Cat#: M2504S). .. For the in vitro acetylation assay, 1 μg of recombinant histone H4 was incubated with 250 ng of purified Esco2-Flag or Esco2-W530G-Flag in 50 μl of acetyltransferase assay buffer (50 mM Tris–HCl pH8.0, 10% glycerol, 10 mM butyric acid, 0.1 mM ethylenediaminetetraacetic acid, 1 mM Dithiothreitol (DTT) and 1mM Phenylmethylsulfonyl fluoride (PMSF) with or without 10 μM Ac-CoA (Sigma) at 30°C for 1 h on a rotating platform.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: .. Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C. .. Bacterial viability was then measured using the LIVE/DEAD Baclight kit (Life Technologies) according to the manufacturer’s protocol.

    Article Title: Histone H4 induces platelet ballooning and microparticle release during trauma hemorrhage
    Article Snippet: .. Washed platelets (3 × 108 /mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. ..

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Activity Assay:

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Histone killing assay To measure the bactericidal activity of histone H4, R . solanacearum GMI1000 cells were grown overnight in CPG, pelleted, washed, and adjusted to an OD600 of 1.5 with sterile water. .. Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C.

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: .. PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Expressing:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: It was preliminarily verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that DNA dose-dependently induced TF mRNA expression in the THP-1 cells (tested at 0, 25 and 50 ng/µl; data not shown) and consequently the highest concentration was used for subsequent experiments. .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.).

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: In vitro acetylation assay Full-length Esco2 and mutant Esco2-W530G cDNAs were sub-cloned into pcDNA3.1/Flag vector, respectively, and then transfected into HEK293 cells for expression, followed by flag purification. .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Western Blot:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. Phosphorylated histone proteins were detected by autoradiography or Western blotting.

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast. .. The gel was processed for Western Blotting using a H4 antibody described previously .

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: Recombinant histone H4 was purchased from NEB (Cat#: M2504S). .. The reaction was then stopped by adding sodium dodecyl sulphate-polyacrylamide gel electrophoresis loading buffer and analyzed by western blotting with anti-H4K16ac antibody.

    Kinase Assay:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: Paragraph title: In vitro kinase assay ... The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S).

    Crystallization Assay:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Purified ORF158L was used for generation of monoclonal antibodies in mouse, crystallization, and protein-binding assays. .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    High Performance Liquid Chromatography:

    Article Title: Pressure-assisted sample preparation for proteomic analysis
    Article Snippet: High-performance liquid chromatography (HPLC) gradient-grade acetonitrile (ACN) and water were purchased from Fisher Scientific (Fair Lawn, NJ, USA). .. Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Transfection:

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: In vitro acetylation assay Full-length Esco2 and mutant Esco2-W530G cDNAs were sub-cloned into pcDNA3.1/Flag vector, respectively, and then transfected into HEK293 cells for expression, followed by flag purification. .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Chromatography:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The recombinant protein was first purified using an Ni-nitrilotriacetic acid (NTA) column (Qiagen) under native conditions and then by gel filtration chromatography using a Superdex 26/75 column (Amersham). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Concentration Assay:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: It was preliminarily verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that DNA dose-dependently induced TF mRNA expression in the THP-1 cells (tested at 0, 25 and 50 ng/µl; data not shown) and consequently the highest concentration was used for subsequent experiments. .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.).

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: .. Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C. .. Bacterial viability was then measured using the LIVE/DEAD Baclight kit (Life Technologies) according to the manufacturer’s protocol.

    Article Title: A screening method for phosphohistidine phosphatase 1 activity
    Article Snippet: It was lyophilized and stored at -20°C until use, when it was dissolved in deionized water to a concentration of 0.2 M. Solutions of phosphoramidate were then stored at -80°C in aliquots that were thawed only once before use. .. Human recombinant histone H4 was from New England Biolabs, UK.

    Cell Culture:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: Paragraph title: Cell culture ... THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.).

    Imaging:

    Article Title: Histone H4 induces platelet ballooning and microparticle release during trauma hemorrhage
    Article Snippet: Washed platelets (3 × 108 /mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. .. Platelets were then prepared for flow cytometry or imaging flow cytometry as described in the SI Appendix .

    Polymerase Chain Reaction:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: It was preliminarily verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) that DNA dose-dependently induced TF mRNA expression in the THP-1 cells (tested at 0, 25 and 50 ng/µl; data not shown) and consequently the highest concentration was used for subsequent experiments. .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Recombinant:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.). .. After 2 h of incubation at 37˚C and 5% CO2 , the THP-1 cells were harvested.

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: .. Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast. .. Reactions were carried out in a total volume of 15µl and incubated for 1h at 30°C, following which 5µl of 4x SDS-PAGE loading buffer was added and the ubiquitylated proteins and the entire reaction was resolved on a 18% SDS-polyacrylamide gel.

    Article Title: Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis *
    Article Snippet: .. Recombinant histone H4 (human) was procured from New England Biolabs. .. Male Wistar rats and female BALB/c mice were obtained from the central animal facility at Indian Institute of Science and animal facility at Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.

    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: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S). .. For the in vitro acetylation assay, 1 μg of recombinant histone H4 was incubated with 250 ng of purified Esco2-Flag or Esco2-W530G-Flag in 50 μl of acetyltransferase assay buffer (50 mM Tris–HCl pH8.0, 10% glycerol, 10 mM butyric acid, 0.1 mM ethylenediaminetetraacetic acid, 1 mM Dithiothreitol (DTT) and 1mM Phenylmethylsulfonyl fluoride (PMSF) with or without 10 μM Ac-CoA (Sigma) at 30°C for 1 h on a rotating platform.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: .. Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C. .. Bacterial viability was then measured using the LIVE/DEAD Baclight kit (Life Technologies) according to the manufacturer’s protocol.

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The recombinant protein was first purified using an Ni-nitrilotriacetic acid (NTA) column (Qiagen) under native conditions and then by gel filtration chromatography using a Superdex 26/75 column (Amersham). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Article Title: Pressure-assisted sample preparation for proteomic analysis
    Article Snippet: .. Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA). .. Primary human monocytes, obtained by elutriation from healthy donors as described in Refs. [ , ], were used to isolate histone fractions using a Qproteome Nuclear Protein Kit (Qiagen, Hilden, Germany).

    Article Title: A screening method for phosphohistidine phosphatase 1 activity
    Article Snippet: .. Human recombinant histone H4 was from New England Biolabs, UK. .. The recombinant protein was delivered, at 1 mg/mL, in 20 mM sodium phosphate (pH 7.0), 300 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol.

    Article Title: Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation
    Article Snippet: .. Materials and methods Recombinant human histone H4 was purchased from NE Biolabs and bovine H3 from Roche. .. Recombinant human TLK1B and Asf1B were prepared as described previously [ ].

    Article Title: Histone H4 induces platelet ballooning and microparticle release during trauma hemorrhage
    Article Snippet: .. Washed platelets (3 × 108 /mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. ..

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: .. PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Nucleic Acid Electrophoresis:

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: Recombinant histone H4 was purchased from NEB (Cat#: M2504S). .. The reaction was then stopped by adding sodium dodecyl sulphate-polyacrylamide gel electrophoresis loading buffer and analyzed by western blotting with anti-H4K16ac antibody.

    Fluorescence:

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C. .. The percentage of live cells was determined by plotting the SYTO9/PI fluorescence ratio against a standard curve based on known mixtures of live and dead cells.

    Methylation:

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: .. PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Mutagenesis:

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: In vitro acetylation assay Full-length Esco2 and mutant Esco2-W530G cDNAs were sub-cloned into pcDNA3.1/Flag vector, respectively, and then transfected into HEK293 cells for expression, followed by flag purification. .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Paragraph title: Gene cloning, site-directed mutagenesis, protein expression, and antibody production. ... Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: .. PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Flow Cytometry:

    Article Title: Histone H4 induces platelet ballooning and microparticle release during trauma hemorrhage
    Article Snippet: Washed platelets (3 × 108 /mL) were recalcified to 2 mM and incubated at 37 °C under stirring conditions with vehicle or Histone H4 Human, Recombinant (New England Biolabs), at the stated concentrations. .. Platelets were then prepared for flow cytometry or imaging flow cytometry as described in the SI Appendix .

    Purification:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: In vitro kinase assay In vitro kinase assays were conducted with 200 ng of purified glutathione S -transferase (GST)–tagged TIE2 active protein (SignalChem, no. T04-11G) or the His-tagged TIE2 with deleted kinase domain (Creative BioMart, no. TEK-153H) in kinase buffer [25 mM MOPS (pH 7.2), 12.5 mM β-glycerol phosphate, 20 mM MgCl2 , 12.5 mM MnCl2, 2 mM EDTA (pH 8.0), 5 mM EGTA (pH 7.0), and 0.25 mM DTT] with the addition of 20 μM adenosine triphosphate (ATP) for cold reactions, and 10 μM ATP mixed with 10 μCi of [γ-32 P]ATP for radioactive reactions. .. The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S).

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: .. Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast. .. Reactions were carried out in a total volume of 15µl and incubated for 1h at 30°C, following which 5µl of 4x SDS-PAGE loading buffer was added and the ubiquitylated proteins and the entire reaction was resolved on a 18% SDS-polyacrylamide gel.

    Article Title: Insights into Role of Bromodomain, Testis-specific (Brdt) in Acetylated Histone H4-dependent Chromatin Remodeling in Mammalian Spermiogenesis *
    Article Snippet: The anti-Brdt IgG was purified from mouse antiserum using Melon Gel IgG purification kit (Pierce) according to manufacturer's instructions. .. Recombinant histone H4 (human) was procured from New England Biolabs.

    Article Title: Human Protein Arginine Methyltransferase 7 (PRMT7) Is a Type III Enzyme Forming \u03c9-NG-Monomethylated Arginine Residues
    Article Snippet: Recombinant human protein complex SmB/D3 purified from E. coli was generously provided by Dr. Christian Kambach (Paul Scherrer Institut, Switzerland) and Dr. Utz Fischer (Theodor-Boveri-Institute, Biocenter, University of Wuerzburg, Germany) and has been previously described ( ). .. 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).

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: In vitro acetylation assay Full-length Esco2 and mutant Esco2-W530G cDNAs were sub-cloned into pcDNA3.1/Flag vector, respectively, and then transfected into HEK293 cells for expression, followed by flag purification. .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects
    Article Snippet: .. HTL treatment in vitro Purified histones, including H2a (M2502S, NEB), H2b (M2505S, NEB), H3 (M2503S, NEB) and H4 (M2504S, NEB) were selected. ..

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The H3/H4 histone complex was purified from vector pET11a-H3H4 as described by English et al. ( ). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Article Title: A screening method for phosphohistidine phosphatase 1 activity
    Article Snippet: Human recombinant phosphohistidine phosphatase (PHPT1) was expressed and purified as described by Ma et al. ( ). .. Human recombinant histone H4 was from New England Biolabs, UK.

    Sequencing:

    Article Title: Pressure-assisted sample preparation for proteomic analysis
    Article Snippet: Molecular-grade dithiothreitol (DTT) and sequencing-grade chymotrypsin were purchased from Promega (Madison, WI, USA). .. Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Acetylation Assay:

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    SDS Page:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S). .. The kinase reaction was stopped by the addition of 4× protein sample buffer (Invitrogen) and boiled for 5 min, followed by SDS-PAGE (16%) analyses.

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast. .. Reactions were carried out in a total volume of 15µl and incubated for 1h at 30°C, following which 5µl of 4x SDS-PAGE loading buffer was added and the ubiquitylated proteins and the entire reaction was resolved on a 18% SDS-polyacrylamide gel.

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. SDS-PAGE gels were fixed with 10% acetic acid/10% methanol v/v, washed, and incubated with Amplify reagent (Amersham) before exposing at −80 °C.

    Plasmid Preparation:

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: In vitro acetylation assay Full-length Esco2 and mutant Esco2-W530G cDNAs were sub-cloned into pcDNA3.1/Flag vector, respectively, and then transfected into HEK293 cells for expression, followed by flag purification. .. Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The H3/H4 histone complex was purified from vector pET11a-H3H4 as described by English et al. ( ). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Negative Control:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.). .. After 2 h of incubation at 37˚C and 5% CO2 , the THP-1 cells were harvested.

    Positron Emission Tomography:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: The orf158L gene was PCR amplified and cloned into pET-15b (Novagen). .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    In Vitro:

    Article Title: TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1
    Article Snippet: Paragraph title: In vitro kinase assay ... The following substrates were used in this study: core histone for calf thymus (Roche, no. 10223565001), histone H3.3 (New England Biolabs, no. M2507S), and histone H4 (New England Biolabs, no. M2504S).

    Article Title: Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis
    Article Snippet: Paragraph title: In vitro ubiquitylation assay ... Where indicated, the reaction was supplemented with the following: 1µg recombinant human histone H4 (New England Biolabs), 1µg recombinant 6-His-tagged human ubiquitin, 0.05µM E1 enzyme Uba1 from yeast, 0.5µM E2 enzyme recombinant GST-UbcH5A (human homolog of yeast Ubc4 which exhibits 81% identity and 94% similarity and can complement the loss of Ubc4 from yeast ) (all from Boston Biochem), 50ng of recombinant 6His-tagged Rad53 and 50ng of Tom1-TAP purified from yeast.

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Article Title: Elevated H3K79 homocysteinylation causes abnormal gene expression during neural development and subsequent neural tube defects
    Article Snippet: .. HTL treatment in vitro Purified histones, including H2a (M2502S, NEB), H2b (M2505S, NEB), H3 (M2503S, NEB) and H4 (M2504S, NEB) were selected. ..

    Article Title: Quantitative reactivity profiling predicts functional cysteines in proteomes
    Article Snippet: .. PRMT1 in vitro methylation assays 500 ng of recombinant human PRMT1 (wild-type or C101A mutant) was pre-incubated with 4-hydroxy-2-nonenal (HNE, Calbiochem) for 30 minutes and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (M2504S; NEB) and 3 H-S-adenosylmethionine (SAM, 2 μCi) in methylation buffer (20 mM Tris [pH 8.0], 200mM NaCl, 0.4 mM EDTA). .. Reactions were incubated for 90 min at 30 °C and stopped with SDS sample buffer.

    Protein Binding:

    Article Title: Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus ▿ †
    Article Snippet: Purified ORF158L was used for generation of monoclonal antibodies in mouse, crystallization, and protein-binding assays. .. Histones H3 (M2507S) and H4 (M2504S) were purchased from New England BioLabs (NEB).

    Activation Assay:

    Article Title: Neutrophil extracellular trap components increase the expression of coagulation factors
    Article Snippet: .. THP-1 cells (500,000 cells per well in 24-well plates) were activated with 50 ng/µl DNA from human neutrophils, or 20 µg/µl human recombinant histone H4 (New England BioLabs, Inc., Ipswich, MA, USA) as described by Kim et al ( ); an equivalent volume of H2 O was used as a negative control and 1 µg/ml Escherichia coli lipopolysaccharide (LPS; 0111:B4; Sigma-Aldrich) was used as a positive control of cell activation, all of which were diluted in Opti-MEM (Thermo Fisher Scientific, Inc.). .. After 2 h of incubation at 37˚C and 5% CO2 , the THP-1 cells were harvested.

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    New England Biolabs recombinant human histone h4
    Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.
    Recombinant Human Histone H4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Time optimization at 15 kpsi for 30, 120, and 200 min for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant

    MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: MALDI–TOF mass spectrum of recombinant human histone H4 digested using the PCT method. The numbered peaks match those generated by conventional chymotrypsin digestion, providing 95% sequence coverage, and are listed in .

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant, Generated, Sequencing

    Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Pressure optimization at 30 min for 10, 15, and 25 kpsi for two peptides from the N-terminal tail portion of recombinant human histone H4.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Recombinant

    RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: RP–HPLC chromatogram showing fractionation of histones using UV detector set at 214 nm. The inset shows an enlarged range of the peptides containing peaks assigned to respective histones.

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: High Performance Liquid Chromatography, Fractionation

    Chymotryptic digestion of native histone H4 using conventional and PCT methods

    Journal: Analytical biochemistry

    Article Title: Pressure-assisted sample preparation for proteomic analysis

    doi: 10.1016/j.ab.2013.03.023

    Figure Lengend Snippet: Chymotryptic digestion of native histone H4 using conventional and PCT methods

    Article Snippet: Recombinant human histone H4 ( ) was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques:

    Esco2 binds to histone H4 to regulate the acetylation level of H4K16. ( A ) Representative images of acetylated H4K16 in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Scale bar, 5 μm. ( B ) The immunofluorescence intensity of acetylated H4K16 was recorded in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a P

    Journal: Nucleic Acids Research

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis

    doi: 10.1093/nar/gkx563

    Figure Lengend Snippet: Esco2 binds to histone H4 to regulate the acetylation level of H4K16. ( A ) Representative images of acetylated H4K16 in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Scale bar, 5 μm. ( B ) The immunofluorescence intensity of acetylated H4K16 was recorded in control, Esco2-KD, Esco2-rescue and Esco2-W530G oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. Asterisk denotes statistical difference at a P

    Article Snippet: Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Techniques: Immunofluorescence

    Esco2 acetylates histone H4 at Lys16 in vitro . ( A ) Flag purification of Esco2. Esco2 and enzymatically mutant Esco2-W530G were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco2-Flag and Esco2-W530G-Flag were detected with sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by both coomassie staining and western blotting with anti-Esco2 antibody. ( B ) Commercially obtained recombinant histone H4 was identified by both coomassie staining and western blotting with anti-H4 antibody. ( C ) In vitro acetylation assay with Esco2-Flag. Recombinant histone H4 was incubated with or without purified Esco2-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control. ( D ) In vitro acetylation assay with Esco2-W530G-Flag. Recombinant histone H4 was incubated with or without purified Esco2-W530G-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis

    doi: 10.1093/nar/gkx563

    Figure Lengend Snippet: Esco2 acetylates histone H4 at Lys16 in vitro . ( A ) Flag purification of Esco2. Esco2 and enzymatically mutant Esco2-W530G were expressed in HEK293 cells and then purified according to the Flag purification procedure. Purified Esco2-Flag and Esco2-W530G-Flag were detected with sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by both coomassie staining and western blotting with anti-Esco2 antibody. ( B ) Commercially obtained recombinant histone H4 was identified by both coomassie staining and western blotting with anti-H4 antibody. ( C ) In vitro acetylation assay with Esco2-Flag. Recombinant histone H4 was incubated with or without purified Esco2-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control. ( D ) In vitro acetylation assay with Esco2-W530G-Flag. Recombinant histone H4 was incubated with or without purified Esco2-W530G-Flag and Ac-CoA in the acetyltransferase assay buffer at 30°C for 1 h. The reactions were analyzed by western blotting with anti-histone H4 (acetyl K16) antibody for acetylation levels of H4K16 and anti-histone H4 antibody as a loading control.

    Article Snippet: Recombinant histone H4 was purchased from NEB (Cat#: M2504S).

    Techniques: In Vitro, Purification, Mutagenesis, Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Recombinant, Acetylation Assay, Incubation

    A. Crosslinking of H3–H4 . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in [ 3 ], before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.

    Journal: BMC Research Notes

    Article Title: Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3-H4 tetramer formation

    doi: 10.1186/1756-0500-2-128

    Figure Lengend Snippet: A. Crosslinking of H3–H4 . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in [ 3 ], before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.

    Article Snippet: Materials and methods Recombinant human histone H4 was purchased from NE Biolabs and bovine H3 from Roche.

    Techniques: Incubation, SDS Page, Western Blot, Staining, Plasmid Preparation

    Dephosphorylation of purified and recombinant histone H4. Histone H4 was, after phosphorylation with phosphoramidate to 1.29 mol/mol, dephosphorylated with 2 pmoles PHPT1 for indicated times. The reaction was performed at 30°C and was interrupted by centrifugation of 50 μL of the reaction mixture through a spin column containing 210 μL DEAE-Sephacel equilibrated in 25 mM Tris/HCl, pH 8.5 at indicated times. Each time point was analysed in duplicate. The phosphate in the final eluate was determined by malachite reagent and histone by absorbance at 280 nm. Details are given in Material and methods.(◊) 40 μM purified phosphohistone H4, and (▪) 20 μM recombinant phosphohistone H4.

    Journal: Upsala Journal of Medical Sciences

    Article Title: A screening method for phosphohistidine phosphatase 1 activity

    doi: 10.3109/03009734.2011.585253

    Figure Lengend Snippet: Dephosphorylation of purified and recombinant histone H4. Histone H4 was, after phosphorylation with phosphoramidate to 1.29 mol/mol, dephosphorylated with 2 pmoles PHPT1 for indicated times. The reaction was performed at 30°C and was interrupted by centrifugation of 50 μL of the reaction mixture through a spin column containing 210 μL DEAE-Sephacel equilibrated in 25 mM Tris/HCl, pH 8.5 at indicated times. Each time point was analysed in duplicate. The phosphate in the final eluate was determined by malachite reagent and histone by absorbance at 280 nm. Details are given in Material and methods.(◊) 40 μM purified phosphohistone H4, and (▪) 20 μM recombinant phosphohistone H4.

    Article Snippet: Human recombinant histone H4 was from New England Biolabs, UK.

    Techniques: De-Phosphorylation Assay, Purification, Recombinant, Centrifugation