recombinant human histone h3 1 protein  (New England Biolabs)


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    Name:
    Histone H3 1 Human Recombinant
    Description:
    Histone H3 1 Human Recombinant 100 ug
    Catalog Number:
    m2503s
    Price:
    82
    Size:
    100 ug
    Category:
    DNA Binding Proteins
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    Structured Review

    New England Biolabs recombinant human histone h3 1 protein
    Histone H3 1 Human Recombinant
    Histone H3 1 Human Recombinant 100 ug
    https://www.bioz.com/result/recombinant human histone h3 1 protein/product/New England Biolabs
    Average 95 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h3 1 protein - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug"

    Article Title: Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

    Journal: Anticancer research

    doi:

    Western blot analysis of histone H3.1 proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).
    Figure Legend Snippet: Western blot analysis of histone H3.1 proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).

    Techniques Used: Western Blot, Methylation

    2) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.
    Figure Legend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Techniques Used: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    3) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.
    Figure Legend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Techniques Used: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    4) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.
    Figure Legend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Techniques Used: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    5) Product Images from "Nuclear Legumain Activity in Colorectal Cancer"

    Article Title: Nuclear Legumain Activity in Colorectal Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052980

    Cleavage of histone H3.1 by active legumain. (A) Immunoblots showing the cleavage of intact (lane 10) recombinant human histone H3.1 in a dose dependent manner by purified mature 36 kDa bovine legumain (bovLeg, lane 1–3) and auto-activated intermediate form (46 kDa) of recombinant human legumain (rhLeg, lane 6–8). The addition of recombinant human cystatin E/M (lane 4 and 8) efficiently blocked legumain activity and resulted in almost complete rescue of histone H3.1 from proteolytic cleavage. Uncut immunoblots ( Fig. S2D ). (B) Immunoblot of histone H3.1 showing the dose-dependent production of a 12 kDa cleavage product after incubation of recombinant histone H3.1 with fully mature 36 kDa bovine legumain in a buffer with pH 7.0 (lane 1–3). Addition of recombinant human cystatin E/M efficiently blocked legumain activity and resulted in virtually no formation of the 12 kDa cleavage product (lane 4).
    Figure Legend Snippet: Cleavage of histone H3.1 by active legumain. (A) Immunoblots showing the cleavage of intact (lane 10) recombinant human histone H3.1 in a dose dependent manner by purified mature 36 kDa bovine legumain (bovLeg, lane 1–3) and auto-activated intermediate form (46 kDa) of recombinant human legumain (rhLeg, lane 6–8). The addition of recombinant human cystatin E/M (lane 4 and 8) efficiently blocked legumain activity and resulted in almost complete rescue of histone H3.1 from proteolytic cleavage. Uncut immunoblots ( Fig. S2D ). (B) Immunoblot of histone H3.1 showing the dose-dependent production of a 12 kDa cleavage product after incubation of recombinant histone H3.1 with fully mature 36 kDa bovine legumain in a buffer with pH 7.0 (lane 1–3). Addition of recombinant human cystatin E/M efficiently blocked legumain activity and resulted in virtually no formation of the 12 kDa cleavage product (lane 4).

    Techniques Used: Western Blot, Recombinant, Purification, Activity Assay, Incubation

    6) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.
    Figure Legend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Techniques Used: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    7) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.
    Figure Legend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Techniques Used: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    8) Product Images from "Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions"

    Article Title: Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv013

    Inclusion of SUV39H2 exon 3 is required to encode an active histone methyltransferase. ( A ) In vitro analysis of the methyltransferase activity associated to each FV G9A and FV SUV39H2 isoforms. Scheme of the in vitro histone methyltransferase (HMT) assay procedure (left panel). Various quantities of purified FV G9A and FV SUV39H2 (right panel) were incubated with the recombinant human histone H3.1 and S-adénosylméthionine (SAM) producing methylated H3.1 and S-adenosylhomocysteine (SAH). Histone H3 and H3K9me3 were analyzed by western blot using specific antibodies (right panel). Control reactions were supplemented with FV Tomato, water (-) or sample issued from blank purification procedure (Ctl). Levels of recombinant proteins were estimated using V5 antibody. ( B ) and ( C ) H3K9me3 was assessed in HeLa cells after expression of FV G9A and FV SUV39H2 isoforms. (B) DNA was counterstained with DAPI (blue), H3K9me3 was immunostained with a specific antibody (red) and cells expressing FV G9A and FV SUV39H2 were revealed with ZsGreen1 Fluorescence Protein (ZsGFP panel; labeled with white arrows in H3K9me3 panel). (C) Analysis of H3K9me3, H3K9me2 and H3 levels by western blot in total protein extracts of HeLa cells expressing FV G9A and FV SUV39H2 isoforms.
    Figure Legend Snippet: Inclusion of SUV39H2 exon 3 is required to encode an active histone methyltransferase. ( A ) In vitro analysis of the methyltransferase activity associated to each FV G9A and FV SUV39H2 isoforms. Scheme of the in vitro histone methyltransferase (HMT) assay procedure (left panel). Various quantities of purified FV G9A and FV SUV39H2 (right panel) were incubated with the recombinant human histone H3.1 and S-adénosylméthionine (SAM) producing methylated H3.1 and S-adenosylhomocysteine (SAH). Histone H3 and H3K9me3 were analyzed by western blot using specific antibodies (right panel). Control reactions were supplemented with FV Tomato, water (-) or sample issued from blank purification procedure (Ctl). Levels of recombinant proteins were estimated using V5 antibody. ( B ) and ( C ) H3K9me3 was assessed in HeLa cells after expression of FV G9A and FV SUV39H2 isoforms. (B) DNA was counterstained with DAPI (blue), H3K9me3 was immunostained with a specific antibody (red) and cells expressing FV G9A and FV SUV39H2 were revealed with ZsGreen1 Fluorescence Protein (ZsGFP panel; labeled with white arrows in H3K9me3 panel). (C) Analysis of H3K9me3, H3K9me2 and H3 levels by western blot in total protein extracts of HeLa cells expressing FV G9A and FV SUV39H2 isoforms.

    Techniques Used: In Vitro, Activity Assay, HMT Assay, Purification, Incubation, Recombinant, Methylation, Western Blot, CTL Assay, Expressing, Fluorescence, Labeling

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    Activity Assay:

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    Expressing:

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    Western Blot:

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    RNA Binding Assay:

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    Transfection:

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
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    Chromatography:

    Article Title: Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2
    Article Snippet: Histone methylation activity assays For ct PRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14 C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins (NEB, M2503S) for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5 mM ZnCl2 , 0.1 mM CaCl2 , 2 mM 2-mercaptoethanol. .. Gel electrophoresis was carried out for 60 min at 150 Volts, and the gel was vacuum dried at 80°C for 30 min on Whatman 3 mm chromatography paper.

    Concentration Assay:

    Article Title: Metabolic interactions between cysteamine and epigallocatechin gallate
    Article Snippet: Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA). .. Briefly, 0.25 μg of EP300 HAT domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl, pH 8.0, 50% glycerol, 0.5 mM EDTA and 5 mM dithiothreitol), 1 μg of histone H3 protein, 10 μM of acetyl-CoA (A2056; Sigma-Aldrich) as well as cysteamine and EGCG (alone or in combination) at the indicated concentration for 1 h at 30°C.

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: Briefly, RNAs were folded as described for binding assays above and were then allowed to bind PRC2 in RNA binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 100 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 and 2 mM 2-mercaptoethanol) at 25 °C for 30 min. Each 10 μL HMTase reaction contained 500 nM PRC2 complex, RNA as indicated throughout the text, 4 μM H3.1 histone protein (NEB M2503S) or 2 μM mononucleosomes (see above for in vitro nucleosome reconstitution), and 5.0 μM S-[methyl- C]-adenosyl-L-methionine (PerkinElmer #NEC363050UC). .. The reactions were then stopped by adding 4× LDS sample buffer (ThermoFisher #NP0007) to a final concentration of 1×.

    Article Title: Nuclear Legumain Activity in Colorectal Cancer
    Article Snippet: Proteolytic cleavage of histone H3.1 Human recombinant legumain (R & D systems, 2199-CY) was auto-activated at 37°C for 2 h in acidic buffer (50 mM NaOAc, 100 mM NaCl, pH 4.0) at concentration 0.1 µg/µl. .. Human recombinant histone H3.1 (New England BioLabs, M2503S) was added to 50 µl assay buffer (50 mM MES, 250 mM NaCl, pH 5.0 or pH 7.0) with or without cystatin E/M and with final addition of either active human or bovine legumain.

    SYBR Green Assay:

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: For ChIP on the 6.6-kb mouse Angptl4 gene, we designed 14 pairs of SYBR Green PCR primers to cover from −10 to +10 kb of the TSS of Angptl4 gene. .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used.

    Cell Culture:

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: 293FT cells cultured in 10 cm dishes were transfected with 10 μg per dish pCMV6-Entry-MLL4 plasmid expressing FLAG-tagged WT or Y5426A/Y5512A mutant full-length human MLL4 using GenJet (SignaGen SL100489). .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot.

    Protein Protein Interaction Assay:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: Paragraph title: Protein–Protein Interaction Assay ... In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500.

    Protein Concentration:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: Protein concentration was determined by the Bradford method using the protein assay kit (Bio-Rad Laboratories), and proteins were stored at −20°C until use. .. In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500.

    Polymerase Chain Reaction:

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: For ChIP on the 6.6-kb mouse Angptl4 gene, we designed 14 pairs of SYBR Green PCR primers to cover from −10 to +10 kb of the TSS of Angptl4 gene. .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used.

    Binding Assay:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: .. Briefly, RNAs were folded as described for binding assays above and were then allowed to bind PRC2 in RNA binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 100 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 and 2 mM 2-mercaptoethanol) at 25 °C for 30 min. Each 10 μL HMTase reaction contained 500 nM PRC2 complex, RNA as indicated throughout the text, 4 μM H3.1 histone protein (NEB M2503S) or 2 μM mononucleosomes (see above for in vitro nucleosome reconstitution), and 5.0 μM S-[methyl- C]-adenosyl-L-methionine (PerkinElmer #NEC363050UC). .. The reactions were incubated for 1 h at 30 °C in HMTase buffer (77.5 mM Tris-HCl pH 8.0 at 30 °C, 155 mM KCl, 3.88 mM MgCl2 , 0.2 mM ZnCl2 , 3.1 mM 2-mercaptoethanol, 0.1 mg/mL BSA (NEB B9000) and 5% v/v glycerol).

    Nucleic Acid Electrophoresis:

    Article Title: Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2
    Article Snippet: Histone methylation activity assays For ct PRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14 C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins (NEB, M2503S) for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5 mM ZnCl2 , 0.1 mM CaCl2 , 2 mM 2-mercaptoethanol. .. Gel electrophoresis was carried out for 60 min at 150 Volts, and the gel was vacuum dried at 80°C for 30 min on Whatman 3 mm chromatography paper.

    Pull Down Assay:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500. .. Histone-enriched nuclear extract was prepared using Arabidopsis plants as described earlier , and the pull-down assay was performed in A300 buffer as described above.

    Fluorescence:

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: Chemical fluorescence signal was activated by ECL + (PerkinElmer), and the image was scanned with Odyssey systems (LI-COR). .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    Methylation:

    Article Title: Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2
    Article Snippet: .. Histone methylation activity assays For ct PRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14 C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins (NEB, M2503S) for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5 mM ZnCl2 , 0.1 mM CaCl2 , 2 mM 2-mercaptoethanol. .. Reactions were then loaded on a Nupage 4–12% Bis-Tris gel (Life Technologies).

    Mutagenesis:

    Article Title: Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2
    Article Snippet: .. Histone methylation activity assays For ct PRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14 C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins (NEB, M2503S) for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5 mM ZnCl2 , 0.1 mM CaCl2 , 2 mM 2-mercaptoethanol. .. Reactions were then loaded on a Nupage 4–12% Bis-Tris gel (Life Technologies).

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: 293FT cells cultured in 10 cm dishes were transfected with 10 μg per dish pCMV6-Entry-MLL4 plasmid expressing FLAG-tagged WT or Y5426A/Y5512A mutant full-length human MLL4 using GenJet (SignaGen SL100489). .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot.

    Isolation:

    Article Title: Nuclear Legumain Activity in Colorectal Cancer
    Article Snippet: Bovine legumain was isolated from kidney as described by Yamane et al. . .. Human recombinant histone H3.1 (New England BioLabs, M2503S) was added to 50 µl assay buffer (50 mM MES, 250 mM NaCl, pH 5.0 or pH 7.0) with or without cystatin E/M and with final addition of either active human or bovine legumain.

    Purification:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: .. In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500. ..

    Article Title: Conserved RNA-binding specificity of polycomb repressive complex 2 is achieved by dispersed amino acid patches in EZH2
    Article Snippet: .. Histone methylation activity assays For ct PRC2, 600 nM of purified wild type or mutant protein was incubated with 12 µM 14 C-labelled SAM (PerkinElmer) and 2.4 µM histone H3.1 proteins (NEB, M2503S) for 30 min in a 1X reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2 , 0.5 mM ZnCl2 , 0.1 mM CaCl2 , 2 mM 2-mercaptoethanol. .. Reactions were then loaded on a Nupage 4–12% Bis-Tris gel (Life Technologies).

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used. .. GST-GCN5 was purified from bacteria and GCN5.com was purified from nuclear extracts of MEFs expressing FLAG-tagged full-length mouse GCN5 as described ( ).

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: DNA was purified from the remaining 60 μl of lysate, and the DNA amount was used as a loading control. .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    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. ..

    Blocking Assay:

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: After blocking by PBS containing 3% BSA, the membrane was incubated with H3 antibody (1:3000–10,000 dilution; ab1791, Abcam) or H4 antibody (1:1000 dilution; ab10158, Abcam) and subsequently with peroxidase-conjugated anti-rabbit antibody (1:4000 dilution; Invitrogen). .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3.

    FACS:

    Article Title: Fetal testis organ culture reproduces the dynamics of epigenetic reprogramming in rat gonocytes
    Article Snippet: Western blot was done with 10 μg of recombinant protein H3 (New England Biolabs, #M2503S, Whitby, Ontario, Canada). .. Representative images of dispersed cells from rat fetal testis before and after FACS sorting.

    Acetylation Assay:

    Article Title: Metabolic interactions between cysteamine and epigallocatechin gallate
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA).

    Article Title: Aspirin Recapitulates Features of Caloric Restriction
    Article Snippet: .. In Vitro Acetylation Assay Recombinant GST-EP300 fusion protein, corresponding to the amino acids 1,066–1,707 (14-418, Millipore), was assessed for its acetyltransferase activity on the EP300 natural substrates recombinant histone H3 protein (M2503S, New England Biolabs). .. Briefly, 1 μg EP300 Histone acetyl transferase (HAT) domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl [pH 8.0], 50% glycerol, 0.5 mM EDTA, and 5 mM dithiothreitol), 1 μg substrate protein, and two different concentrations of AcCoA (A2056, Sigma-Aldrich) for 1 hr at 30°C in the presence of AA, C646, and sodium salicylate.

    Article Title: Spermidine induces autophagy by inhibiting the acetyltransferase EP300
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA).

    Chromatin Immunoprecipitation:

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: Paragraph title: Western blot, qRT–PCR, ChIP and in vitro HAT assay ... In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used.

    SDS Page:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500. .. The amount of histone H3 bound to the beads was determined by SDS-PAGE and Coomassie Brilliant Blue staining or immunoblotting.

    Article Title: Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii
    Article Snippet: Immunoprecipitated HA TgGCN5b (see above) or recombinant human P300 (catalogue number 14-418; Millipore) was incubated with a recombinant human histone H3.1 substrate (catalogue number M2503S; New England BioLabs) and acetyl coenzyme A (acetyl-CoA) (catalogue number A2056; Sigma) in KAT buffer (50 mM Tris-Cl [pH 8.0], 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 10 mM sodium butyrate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 1 h at 37°C. .. KAT reaction mixtures were then resolved by 4 to 12% Bis-Tris SDS-PAGE (Life Technologies).

    Plasmid Preparation:

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: 293FT cells cultured in 10 cm dishes were transfected with 10 μg per dish pCMV6-Entry-MLL4 plasmid expressing FLAG-tagged WT or Y5426A/Y5512A mutant full-length human MLL4 using GenJet (SignaGen SL100489). .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot.

    Real-time Polymerase Chain Reaction:

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: The sequences of quantitative PCR primers are listed in . .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used.

    Recombinant:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: .. In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500. ..

    Article Title: Fetal testis organ culture reproduces the dynamics of epigenetic reprogramming in rat gonocytes
    Article Snippet: .. Western blot was done with 10 μg of recombinant protein H3 (New England Biolabs, #M2503S, Whitby, Ontario, Canada). .. Membranes were incubated with anti-H3 (1/10,000) (A), anti-H3K4me2 (1/2000) (B) and anti-H3K4me3 (1/2000) (C).

    Article Title: Metabolic interactions between cysteamine and epigallocatechin gallate
    Article Snippet: .. Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA). .. Briefly, 0.25 μg of EP300 HAT domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl, pH 8.0, 50% glycerol, 0.5 mM EDTA and 5 mM dithiothreitol), 1 μg of histone H3 protein, 10 μM of acetyl-CoA (A2056; Sigma-Aldrich) as well as cysteamine and EGCG (alone or in combination) at the indicated concentration for 1 h at 30°C.

    Article Title: Aspirin Recapitulates Features of Caloric Restriction
    Article Snippet: .. In Vitro Acetylation Assay Recombinant GST-EP300 fusion protein, corresponding to the amino acids 1,066–1,707 (14-418, Millipore), was assessed for its acetyltransferase activity on the EP300 natural substrates recombinant histone H3 protein (M2503S, New England Biolabs). .. Briefly, 1 μg EP300 Histone acetyl transferase (HAT) domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl [pH 8.0], 50% glycerol, 0.5 mM EDTA, and 5 mM dithiothreitol), 1 μg substrate protein, and two different concentrations of AcCoA (A2056, Sigma-Aldrich) for 1 hr at 30°C in the presence of AA, C646, and sodium salicylate.

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used. .. GST-GCN5 was purified from bacteria and GCN5.com was purified from nuclear extracts of MEFs expressing FLAG-tagged full-length mouse GCN5 as described ( ).

    Article Title: Mapping of histone-binding sites in histone replacement-completed spermatozoa
    Article Snippet: .. Recombinant proteins H3.1 (M2503S, NEB) and H3.3 (M2507S, NEB) were analyzed for checking sensitivities of H3 antibody to H3.1 and H3.3. ..

    Article Title: Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2
    Article Snippet: .. Some experiments were conducted in the presence of recombinant histone H3.1 (Cat. No. M2503S, New England Biolabs), as detailed in the ‘Results‘ section. .. Co-immunoprecipitation assay For co-immunoprecipitation of DNMT3A and MeCP2, pcDNA-DNMT3A (expressing myc tagged DNMT3A) and pEYFP-MeCP2 plasmids were co-transfected in HEK293 cells.

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot. .. To generate MLL4 enzyme-dead KI ES cells, the linearized gene targeting construct ( ) was electroporated into wild type ES cells.

    Article Title: Spermidine induces autophagy by inhibiting the acetyltransferase EP300
    Article Snippet: .. Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA). .. Briefly, 1 μ g of EP300 HAT domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl, pH 8.0, 50% glycerol, 0.5 mM EDTA and 5 mM dithiothreitol), 1 μ g of histone H3 protein and 10 or 100 μ M of acetyl-CoA (A2056; Sigma-Aldrich) for 1 h at 30 °C.

    Article Title: Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii
    Article Snippet: .. Immunoprecipitated HA TgGCN5b (see above) or recombinant human P300 (catalogue number 14-418; Millipore) was incubated with a recombinant human histone H3.1 substrate (catalogue number M2503S; New England BioLabs) and acetyl coenzyme A (acetyl-CoA) (catalogue number A2056; Sigma) in KAT buffer (50 mM Tris-Cl [pH 8.0], 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 10 mM sodium butyrate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 1 h at 37°C. .. KAT reaction mixtures were then resolved by 4 to 12% Bis-Tris SDS-PAGE (Life Technologies).

    Article Title: Nuclear Legumain Activity in Colorectal Cancer
    Article Snippet: .. Human recombinant histone H3.1 (New England BioLabs, M2503S) was added to 50 µl assay buffer (50 mM MES, 250 mM NaCl, pH 5.0 or pH 7.0) with or without cystatin E/M and with final addition of either active human or bovine legumain. .. Legumain and cathepsin L are heterogeneously expressed in CRC cell lines Lysates from a panel of CRC cell lines were subjected to separation by PAGE and blotted onto PVDF membranes.

    In Vitro:

    Article Title: Metabolic interactions between cysteamine and epigallocatechin gallate
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA).

    Article Title: Aspirin Recapitulates Features of Caloric Restriction
    Article Snippet: .. In Vitro Acetylation Assay Recombinant GST-EP300 fusion protein, corresponding to the amino acids 1,066–1,707 (14-418, Millipore), was assessed for its acetyltransferase activity on the EP300 natural substrates recombinant histone H3 protein (M2503S, New England Biolabs). .. Briefly, 1 μg EP300 Histone acetyl transferase (HAT) domain was incubated in the presence of an HAT assay buffer (250 mM Tris-HCl [pH 8.0], 50% glycerol, 0.5 mM EDTA, and 5 mM dithiothreitol), 1 μg substrate protein, and two different concentrations of AcCoA (A2056, Sigma-Aldrich) for 1 hr at 30°C in the presence of AA, C646, and sodium salicylate.

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: .. Briefly, RNAs were folded as described for binding assays above and were then allowed to bind PRC2 in RNA binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 100 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 and 2 mM 2-mercaptoethanol) at 25 °C for 30 min. Each 10 μL HMTase reaction contained 500 nM PRC2 complex, RNA as indicated throughout the text, 4 μM H3.1 histone protein (NEB M2503S) or 2 μM mononucleosomes (see above for in vitro nucleosome reconstitution), and 5.0 μM S-[methyl- C]-adenosyl-L-methionine (PerkinElmer #NEC363050UC). .. The reactions were incubated for 1 h at 30 °C in HMTase buffer (77.5 mM Tris-HCl pH 8.0 at 30 °C, 155 mM KCl, 3.88 mM MgCl2 , 0.2 mM ZnCl2 , 3.1 mM 2-mercaptoethanol, 0.1 mg/mL BSA (NEB B9000) and 5% v/v glycerol).

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used. .. GST-GCN5 was purified from bacteria and GCN5.com was purified from nuclear extracts of MEFs expressing FLAG-tagged full-length mouse GCN5 as described ( ).

    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: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot. .. To generate MLL4 enzyme-dead KI ES cells, the linearized gene targeting construct ( ) was electroporated into wild type ES cells.

    Article Title: Spermidine induces autophagy by inhibiting the acetyltransferase EP300
    Article Snippet: Paragraph title: In vitro acetylation assay ... Recombinant GST-EP300 fusion protein, corresponding to amino acids 1066–1707 (14–418; Millipore, Billerica, MA, USA) was assessed for its acetyltransferase activity on the EP300 natural substrate recombinant histone H3 protein (M2503S; New England Biolabs, Ipswich, MA, USA).

    Article Title: Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii
    Article Snippet: Paragraph title: In vitro lysine acetyltransferase assays. ... Immunoprecipitated HA TgGCN5b (see above) or recombinant human P300 (catalogue number 14-418; Millipore) was incubated with a recombinant human histone H3.1 substrate (catalogue number M2503S; New England BioLabs) and acetyl coenzyme A (acetyl-CoA) (catalogue number A2056; Sigma) in KAT buffer (50 mM Tris-Cl [pH 8.0], 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 10 mM sodium butyrate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 1 h at 37°C.

    Quantitation Assay:

    Article Title: Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation
    Article Snippet: PCR quantitation of precipitated genomic DNA relative to inputs was performed in duplicate or triplicate using SYBR Green kit. .. In vitro HAT assays were performed on 1 μg recombinant histone H3.1 (New England Biolabs, M2503S) in the presence of acetyl CoA as described , except that 1.5 μg recombinant GST-GCN5 or 1.0 μg GCN5-associated HAT complexes (GCN5.com) were used.

    Immunoprecipitation:

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: MLL4-associated proteins were immunoprecipitated from nuclear extracts using anti-FLAG M2 agarose, followed by elution with the FLAG peptide as described [ ]. .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot.

    Article Title: Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii
    Article Snippet: .. Immunoprecipitated HA TgGCN5b (see above) or recombinant human P300 (catalogue number 14-418; Millipore) was incubated with a recombinant human histone H3.1 substrate (catalogue number M2503S; New England BioLabs) and acetyl coenzyme A (acetyl-CoA) (catalogue number A2056; Sigma) in KAT buffer (50 mM Tris-Cl [pH 8.0], 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 10 mM sodium butyrate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 1 h at 37°C. .. KAT reaction mixtures were then resolved by 4 to 12% Bis-Tris SDS-PAGE (Life Technologies).

    Staining:

    Article Title: A WD40 Domain Cyclophilin Interacts with Histone H3 and Functions in Gene Repression and Organogenesis in Arabidopsis
    Article Snippet: In the assay, 1.5 μg of purified recombinant GST-CYP71 or GST was immobilized to glutathione–Sepharose beads that were incubated with 15 μg of core histones (13-107; Upstate) or 0.5 μg of recombinant histone H3 peptide (M2503S; New England Biolabs) for 6 or 1 h at 4°C in 400 μL of buffer A300 or A500. .. The amount of histone H3 bound to the beads was determined by SDS-PAGE and Coomassie Brilliant Blue staining or immunoblotting.

    Article Title: Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2
    Article Snippet: Proteins were detected by western blotting or Coomassie BB staining as indicated. .. Some experiments were conducted in the presence of recombinant histone H3.1 (Cat. No. M2503S, New England Biolabs), as detailed in the ‘Results‘ section.

    HMT Assay:

    Article Title: H3K4 methyltransferase activity is required for MLL4 protein stability
    Article Snippet: .. For the in vitro HMT assay, 1 μg of MLL4-associated proteins were incubated with 1 μg of recombinant histone H3.1 (NEB M2503S) in the HMT buffer (50 mM Tris pH 8.5, 100 mM KCl, 5 mM MgCl2 , 10% glycerol, 4 mM DTT) supplemented with S-adenosyl methionine (NEB, B9003S) at 37 °C for 2 h, followed by Western blot. .. To generate MLL4 enzyme-dead KI ES cells, the linearized gene targeting construct ( ) was electroporated into wild type ES cells.

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    New England Biolabs recombinant human histone h3 1 protein
    Western blot analysis of histone <t>H3.1</t> proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).
    Recombinant Human Histone H3 1 Protein, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human histone h3 1 protein/product/New England Biolabs
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    Western blot analysis of histone H3.1 proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).

    Journal: Anticancer research

    Article Title: Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

    doi:

    Figure Lengend Snippet: Western blot analysis of histone H3.1 proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).

    Article Snippet: Recombinant human histone H3.1 protein (MW=15273.2 Da by ESI-TOF) was purchased from New England BioLabs, Inc. (Ipswich, MA, USA).

    Techniques: Western Blot, Methylation

    The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Journal: The Journal of nutritional biochemistry

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: The N-, C-, and the linker domains in human HCS interact with histone H3.2 in yeast-two-hybrid assays (A) plate layout of HCS interactions; the interaction between p53 and T antigen was used as positive control; (B) activation of reporter genes and secretion of a-galactosidase, mediated by HCS-H3 interactions (arrows); and (C) successful co-transformation of test plasmids was verified by growing AH109 host strain on SD/−Leu, −Trp, +Kan plates;.

    Article Snippet: Input control and recombinant human histone H3.2 (NewEngland Biolabs, Ipswich, MA) were used as positive controls, whereas the sample precipitated with non-specific IgG was used as negative control.

    Techniques: Positive Control, Activation Assay, Transformation Assay

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Journal: The Journal of nutritional biochemistry

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Article Snippet: Input control and recombinant human histone H3.2 (NewEngland Biolabs, Ipswich, MA) were used as positive controls, whereas the sample precipitated with non-specific IgG was used as negative control.

    Techniques: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Journal: The Journal of nutritional biochemistry

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Article Snippet: Input control and recombinant human histone H3.2 (NewEngland Biolabs, Ipswich, MA) were used as positive controls, whereas the sample precipitated with non-specific IgG was used as negative control.

    Techniques: Recombinant, Incubation, Generated, Staining