The purified nucleosome (0.38 μM), containing H3.1 or CENP-A, was incubated with trypsin (2.5, 5.0, 7.5, 10 ng, purchased from Sigma-Aldrich) in 10 µl of reaction solution, containing 8 mM Tris-HCl (pH 7.5), 0.5 mM MgCl2, and 1 mM DTT, at 25 °C for 1 min. The reaction was stopped by adding 10 µl of a 4% SDS solution, containing 0.10 M Tris-HCl (pH 6.8), 20% glycerol, and 0.2% bromophenol blue, and the samples were boiled at 95 °C for 15 min. For the western blotting, 6.0 µl portions of the samples were analyzed by 18% SDS-PAGE at 200 V for 100 min. After SDS-PAGE, the western blotting analyses were performed by the method described above, using an anti-H4 rabbit polyclonal antibody (1:1000; Abcam, ab7311) and an HRP-anti rabbit-IgG F(ab’)2 fragment (1:5000; GE Healthcare, NA9340) as the primary and secondary antibodies, respectively. The blocking, primary antibody reaction, and secondary antibody reaction were performed at 4 °C overnight, 4 °C for 3–12 h, and 4 °C for 1–2 h, respectively. The membrane was washed with PBS-T, and then treated with Amersham ECL Prime (GE Healthcare). Chemiluminescence was detected with an LAS4000 image analyzer (GE Healthcare). The uncropped images of all blots are presented in Supplementary information. The uncropped images of all blots are presented in Supplementary Figure 11 .
Ab7311
Manufactured by Abcam
Ab7311 is a monoclonal antibody that can be used for the detection of the target protein. The antibody recognizes and binds to the target protein, which can be useful in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (3 mentions)
Anti h3k9ac 61251, by Active Motif (2 mentions)
Anti h3k18ac 39755, by Active Motif (2 mentions)
Anti gst sc 459 antibody, by Santa Cruz Biotechnology (2 mentions)
Fluorescent secondary antibodies 926 32211, by LI COR (2 mentions)
Mononucleosomes, by EpiCypher (2 mentions)
Anti flag m2 antibody f1804, by Merck Group (2 mentions)
Gateway technique, by Thermo Fisher Scientific (2 mentions)
Anti acetyl histone h4 antibody, by Merck Group (2 mentions)
Pgex 6p 1 vector, by GE Healthcare (2 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Las 4000 image analyzer, by GE Healthcare (1 mentions)
Trypsin, by Merck Group (1 mentions)
Anti h4, by Abcam (1 mentions)
Anti jarid2, by Novus Biologicals (1 mentions)
Anti atrx, by Cell Signaling Technology (1 mentions)
Anti ha, by Roche (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Ab8898, by Abcam (1 mentions)
Clone dm1α, by Merck Group (1 mentions)
12 protocols using ab7311
1
Histone Variant Nucleosome Proteolysis
2
Dinucleosome-Protein Interaction Assay
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WT and uH2A dinucleosomes were incubated with prewashed, magnetic, streptavidin coated beads (Fisher) for 1 h at 4 °C, and then washed five times with 250 mM NaCl, 10 mM Tris-HCl pH 7.0 and 0.01% Tween 20. 10 μg dinucleosomes (WT or uH2A) were incubated with 250 ug nuclear cell extract for 1 h at 4 °C, again washed five times with 250 mM NaCl, 10 mM Tris-HCl pH 7.0 and 0.01% Tween 20, and finally the reaction stopped by adding SDS loading buffer. Samples were analysed by SDS-PAGE and western blot. Primary antibodies used were anti-HA (Roche 3F10 11867423001) at 1:1,000 dilution, anti-ATRX (a gift from R.Gibbons) at 1:10 dilution, anti-EZH2 (Cell Signalling 5246) at 1:1,000 dilution, anti-JARID2 (Novus Biologicals NB100-2214) at 1:1,000 dilution and anti-H4 (Abcam AB7311).
3
Endogenous SUV39H1 Detection in DLD-1 Cells
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Rabbit anti-MBP, anti-GFP, and anti-HP1α polyclonal antibodies were generated by Cocalico Biologicals and purified from rabbit serum. All primary antibodies were incubated for 1 hr (except for anti-SUV39H1 which was incubated overnight) at the following dilutions: rabbit anti-MBP (Straight lab, 0.2 μg/mL), rabbit anti-GFP (Straight lab, 1 μg/mL), rabbit anti-HP1α (Straight lab, 1 μg/mL), mouse anti-SUV39H1 (Millipore clone MG44, cat. #05–615, 1:500), rabbit anti-H3K9me3 (AbCam ab8898, 1:1000), rabbit anti-histone H4 (AbCam ab7311, 1:2000), and mouse monoclonal anti-tubulin (clone DM1α, Sigma T6199, 0.5 μg/mL).
To western blot for endogenous SUV39H1 in DLD-1 cells, nuclear lysates were prepared to enrich for the SUV39H1 containing fraction. Cells were incubated in hypotonic buffer (10 mM Tris-HCl pH 8, 1.5 mM MgCl2, 10 mM KCl) for 10 min on ice, dounced 10 times with a Kontes glass dounce (B pestle), nuclei were pelleted at 5000xg for 10 min, then lysed in DLB (Singh et al., 2014 (link)). If necessary, lysates were sonicated with a Branson Sonifier S-250A using a microtip until no longer viscous. To test the effect of RNase A on SUV39H1 protein levels, pelleted nuclei were incubated with 0.5 mg/mL RNase A, ±4 U/μL RNaseOUT, for 30 min before lysis.
To western blot for endogenous SUV39H1 in DLD-1 cells, nuclear lysates were prepared to enrich for the SUV39H1 containing fraction. Cells were incubated in hypotonic buffer (10 mM Tris-HCl pH 8, 1.5 mM MgCl2, 10 mM KCl) for 10 min on ice, dounced 10 times with a Kontes glass dounce (B pestle), nuclei were pelleted at 5000xg for 10 min, then lysed in DLB (Singh et al., 2014 (link)). If necessary, lysates were sonicated with a Branson Sonifier S-250A using a microtip until no longer viscous. To test the effect of RNase A on SUV39H1 protein levels, pelleted nuclei were incubated with 0.5 mg/mL RNase A, ±4 U/μL RNaseOUT, for 30 min before lysis.
4
Chromatin Immunoprecipitation Antibody Panel
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The purified rabbit polyclonal carboxy terminus-specific anti-Ikaros antibody and the mouse monoclonal anti-ER and anti-Cre antibodies were generated in-house. The N terminus-specific anti-Ikaros (sc-13039, Santa Cruz), anti-Suz12 (3737, Cell Signaling; sc46264, Santa Cruz), anti-Ezh2 (3147S, Cell Signaling), anti-Mta2 (ab8106, Abcam), anti-Mi2β (CHD4; ab70469, Abcam), anti-H3K27me3 (07-449, Millipore), anti-H3K4me3 (ab8580, Abcam), anti-H3 (06-755, Millipore; ab1791 Abcam), anti-H4 (ab7311, Abcam) and anti-β-actin (A5441; Sigma) antibodies were purchased.
5
Histone Variant Detection by Western Blot
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Isolated histones were separated on a 4 to 12% NuPAGE bis-tris gel and transferred to a 0.2-μm nitrocellulose blotting membrane. Membranes were blocked in TBS-T [50 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] containing 5% milk powder and probed for H3.3 (Thermo Fisher Scientific, MA5-24667; 1:5000), H3 (Abcam, ab1791; 1:5000), and H4 (Abcam, ab7311; 1:5000) primary antibodies diluted in TBS-T containing 5% milk powder. This was followed by anti-rabbit horseradish peroxidase (Abcam, ab99697; 1:10,000) and visualized using enhanced chemiluminesence prime Western blotting detection chemiluminescent reagent.
6
Cloning and Characterization of p300 Domains
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The coding DNA sequences (CDS) encoding human full-length p300 protein and the p300 BD-RING-PHD-HAT-ZZ-TAZ2 region (BRPHZT, amino acids 1035–1830) were cloned into pENTR3C vector and subsequently cloned into p3FLAG and pCDH-FLAG destination vectors using Gateway techniques (Invitrogen), respectively. The CDS encoding the human p300 BD-RING-PHD-HAT-ZZ region (BRPHZ, amino acids 1035–1720) and ZZ domain (amino acids 1650–1720) were cloned into the pGEX-6P-1 vector (GE Healthcare). Point mutations and deletions were generated using a site-directed mutagenesis kit (Stratagene) and verified by Sanger sequencing. Histone peptides bearing different modifications were synthesized at CPC, LLC. Anti-histone antibodies including anti-H3 (Ab1791), anti-H3K4ac (ab176799), anti-H3K9ac (Ab32129), anti-H3K27ac (Ab4729) and anti-H4 (Ab7311) antibodies were obtained from Abcam. Anti-H3K9ac (61251) and anti-H3K18ac (39755) antibodies were from Active Motif. Anti-acetyl-Histone H4 antibody (06–598) was from Millipore. Anti-GST (sc-459) antibody was from Santa Cruz. Anti-FLAG (M2, F1804) antibody was from Sigma. Fluorescent secondary antibodies (926–32211 and 926–68020) were from LI-COR. Mononucleosomes reconstituted from recombinant histones (16–0009) were from EpiCypher.
7
Quantitative Analysis of Histone Acetylation
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In total, 100 nM H3K9ac or H3K27ac nucleosomes (Active Motif) were incubated on ice with 100 nM protein in buffer containing 25 mM HEPES pH 7.5, 50 mM NaCl, and 0.1 mM TCEP. Reactions were stopped at various timepoints by quenching with gel loading buffer (NuPAGE LDS sample buffer, Life Technologies), loaded onto 4–12% SDS-PAGE gels and transferred onto PVDF membranes for western blotting using site-specific primary antibodies, H3K9ac, H3K27ac, H3K14ac and H4 (Cell Signalling #9649, #D5E4, Sigma-Aldrich #07-353, and Abcam #ab7311 respectively). Membranes were subsequently probed with goat-anti-rabbit-HRP conjugated secondary antibody (Abcam, #ab205718) then incubated with ECL for detection and quantified using ImageJ Fiji v2.1.0/1.53c. All the primary antibodies were diluted 1:2000. The secondary antibody was diluted 1:10,000.
8
Yeast Strain Generation and Characterization
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Yeast strains were grown under standard conditions, and gene deletion and tagging were performed as described previously (84 (link)). The list of yeast strains and plasmids used for various studies is provided in Table S1 and S2 , respectively. Plasmid transformations were performed as described previously (85 (link)). Primers used for deletion and tagging genes are listed in Table S3 . Genomic integration of the tagged and the deleted genes was confirmed by PCR. Antibodies used were as follows: anti-HA antibody, ChIP grade (ab9110, Abcam), anti-histone H4 antibody, ChIP grade (ab7311, Abcam), anti-histone H4Ac pan-acetyl antibody, ChIP grade (39243, Active Motif), H2A (1:5000; 39325, Active Motif), H2ASer129Ph (39271, Active Motif), anti-Rad53 (ab104232, Abcam), G6PDH (1:100,000; A9521, Sigma-Aldrich), and rabbit (Amersham NA934; donkey anti-rabbit) and mouse (Amersham NA931; sheep anti-mouse) secondary antibodies were used at 1:10,000.
9
ASXL2 Histone Methyltransferase Assay
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Equivalent amounts of purified ASXL2 WT and ΔMBH complexes were incubated with 0.5 μg of reconstituted recombinant mononucleosomes or histone octamers as substrates 42 . All HMT assays were performed in 50 mM Tris-HCl pH 8, 100 mM KCl, 5 mM MgCl 2 , 0.1 mM DTT, 10% glycerol in the presence of 50 μM S-adenosyl-Lmethionine (Sigma, A7007). After an 8 h incubation at 30 °C, 4X Laemmli buffer was added, the samples run on 15% SDS-PAGE and transferred on nitrocellulose membrane. Immunoblotting was performed with an anti-H3K4me1 (Ab8895, rabbit polyclonal) diluted 1:1000 followed by HRP conjugated secondary goat anti-rabbit antibody (Sigma) used at a 1:10,000 dilution. The immunoblots were visualized using a Western Lightning plus-ECL reagent (Perkin-Elmer). Anti-H4 (Abcam, Ab7311, rabbit polyclonal) was used at a 1:2,500 dilution.
10
Quantifying Centromeric Histone CENH3
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Approximately 2 g of flash-frozen leaves or roots were collected and chopped into 1.5 ml pre-chilled nuclei extraction buffer (1mM EDTA, 1x cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 5 mM 2-mercaptoethanol, 0.1 mM PMSF). The mixture was poured through miracloth and filtered through a 40 m cell strainer. Then, 30 l of the filtered sample was stained with 4,6-diamidino-2-phenylindole and nuclei counted using fluorescence microscopy. Nuclei concentrations were normalized based on these measurements. The nuclei were centrifuged at 5000 g for 5 mins and the pellets flashfrozen and stored at -80℃ until used for protein blots. Nuclei were resuspended in Laemmli buffer and loaded into 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad Cat #4561093). SDS-PAGE and protein blotting were performed according to (Dawe et al. 2018) .
CENH3 was detected with anti-CENH3 antibodies (Zhong et al. 2002) (1:1000 dilution) and normalized to total H4 histones revealed by an anti-H4 antibodies (1:1000 dilution, Abcam, ab7311). Primary antibodies were detected using anti-rabbit secondary antibodies (1:5000 dilution, Anti-Rabbit IgG HRP Linked Whole Ab Sigma Cat# GENA934-1ML). The band intensities were quantified with Image J (Schneider et al. 2012) .
CENH3 was detected with anti-CENH3 antibodies (Zhong et al. 2002) (1:1000 dilution) and normalized to total H4 histones revealed by an anti-H4 antibodies (1:1000 dilution, Abcam, ab7311). Primary antibodies were detected using anti-rabbit secondary antibodies (1:5000 dilution, Anti-Rabbit IgG HRP Linked Whole Ab Sigma Cat# GENA934-1ML). The band intensities were quantified with Image J (Schneider et al. 2012) .
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