To extract histone proteins, embryos dissected at the indicated embryonic days, or about 5 × 106 cells (were lysed with 100 μl acidic extraction buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.2 M HCl) freshly complemented with 1 × Proteinase Inhibitor Cocktail (Roche) and 10 mM N-ethylmaleimide (Sigma-Aldrich). HCl was added to a final concentration of 0.2 M and incubated on an end-to-end rotator for 2 h at 4 °C. Following the incubation, cell extract was centrifuged at 20,800 × g for 10 min at 4 °C, to pellet the acid-insoluble material. A solution of 2 M Tris-HCl (pH 8.8) was added to neutralize the supernatant of the acidic extraction. Ten μl of the supernatant, containing histone proteins, were run on 4–12% gels (Bis-tris NuPAGE Novex, Life Technologies), then proteins were transferred and western blot assays were carried out by using standard methods. The following antibodies were used: anti-H3 (Abcam #ab1791) anti-H4 (Invitrogen 3HH4–4G8), anti-H2Bub1 (Cell Signaling Technology, #5546), anti-H3K4me3 (Abcam ab8580), anti-H3K9ac (Merck-Millipore #07-352), Peroxidase AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (Jackson ImmunoResearch #115-036-071) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch #115-035-144). Protein levels were quantified by ImageJ.
Anti h2bub1
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Anti-H2Bub1 is a laboratory reagent used for the detection and analysis of histone H2B ubiquitination. It is a specific antibody that recognizes the ubiquitinated form of histone H2B, a post-translational modification involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (2 mentions)
Bis tris nupage novex, by Thermo Fisher Scientific (2 mentions)
Anti h3k9ac, by Merck Group (2 mentions)
Anti h3k4me3, by Abcam (2 mentions)
Anti h3, by Abcam (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Proteinase inhibitor cocktail, by Roche (2 mentions)
Anti h3k4me3, by Merck Group (1 mentions)
Anti c myc 9e10, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Sc 390585, by Santa Cruz Biotechnology (1 mentions)
Magna chip protein a magnetic beads, by Merck Group (1 mentions)
Anti trimethyl h3k4, by Abcam (1 mentions)
Bioruptor plus sonication device, by Diagenode (1 mentions)
5 protocols using anti h2bub1
1
Histone Extraction from Embryonic Cells
2
Antibodies for Western Blot and ChIP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used in this study for the western blot and the ChIP assay: anti-H3K4me3 (Millipore Cat# 07–473, RRID: AB_1977252; 1/50,000 dilution for WB; 1 μl was added to a chromatin extract for ChIP), anti-H2Bub1 (Cell Signaling Technology Cat# 5546, RRID:AB_10693452; 1/2500 dilution for WB), anti-c-Myc (9E10) (Santa Cruz Biotechnology Cat# sc-40, RRID:AB_627268; 1/5000 dilution for WB; 10 μl was added to a chromatin extract for ChIP), and anti-HA (Santa Cruz Biotechnology Cat# sc-7392, RRID:AB_627809; 1/5000 dilution for WB; 2.5 μl was added to a chromatin extract for ChIP). Anti-H3 (1/100,000 dilution for WB), anti-H3K4me2 (1/50,000 dilution for WB), and anti-Set1(1/5000 dilution for WB; 2.5 μl was added to a chromatin extract for ChIP) were obtained from Shilatifard’s laboratory [15 (link), 17 (link)].
3
Western Blot Analysis of USP22 Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blot analyses, control and USP22-depleted HCT116 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS) containing protease, phosphatase and deubiquitinase inhibitors (1 ng/μL aprotinin/leupeptin, 10 mM β-glycerophosphate, 1 mM N-ethylmaleimide, 1 mM Pefabloc). Upon sonication for 15 min, proteins were denatured in 6× Laemmli buffer at 95 °C for 5 min, separated by SDS-PAGE and blotted onto nitrocellulose membranes. The following antibodies were used: anti-H2B (ab1790, Abcam, Cambridge, UK, 1:5000), anti-H2Bub1 (5546, Cell Signaling, Danvers, MA, USA, 1:1000), anti-USP22 (sc-390585, Santa Cruz, Dallas, TX, USA, 1:1000).
4
Chromatin Immunoprecipitation in Plants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP experiments were performed as previously described (Saleh et al., 2008 ) with some modifications. A 3 g sample of leaves was fixed with cross‐linking buffer and 1% formaldehyde using vacuum infiltration two times for 15 min each, and the cross‐linking reaction was quenched with 0.125 m glycine. The leaves were ground in a mortar and pestle in liquid nitrogen, resuspended in nuclei isolation buffer and then filtered through Miracloth. After centrifugation, the pellets (nuclei) were resuspended in cold nuclei lysis buffer and sonicated (Bioruptor® Plus sonication device, Diagenode, Belgium). After centrifugation, the supernatant was pre‐cleared with Magna ChIP™ Protein A Magnetic Beads (EMD Millipore corporation, Temecula, CA), and specific antibodies were added and incubated overnight at 4 °C. The specific antibodies used were as follows: anti‐H2Bub1 (Cell Signaling Technology, Danvers, MA) and anti‐trimethyl‐H3K4 (Abcam). The enriched DNA fragments were detected by RT‐qPCR and compared with the input samples, the amount of immunoprecipitated chromatin as normalized to the total amount of chromatin used in GhDREB P1 region in wild‐type plants was given as 1. GhUBI1 was used as a negative control.
5
Histone Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract histone proteins, embryos dissected at the indicated embryonic days, or about 5 x10 6 cells (were lysed with 100 μl acidic extraction buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT and 0.2 M HCl) freshly complemented with 1× Proteinase Inhibitor Cocktail (Roche) and 10 mM N-ethylmaleimide (Sigma-Aldrich).
HCl was added to a final concentration of 0.2 M and incubated on an end-to-end rotator for 2 hours at 4°C. Following the incubation, cell extract was centrifuged at 20 800 x g for 10 min at 4°C, to pellet the acid insoluble material. A solution of 2 M Tris-HCl (pH 8.8) was added to neutralize the supernatant of the acidic extraction. Ten μl of the supernatant, containing histone proteins, were run on 4-12% gels (Bis-tris NuPAGE Novex, Life Technologies), then proteins were transferred and western blot assays were carried out by using standard methods. The following antibodies were used: anti-H3 (Abcam #ab1791) anti-H4 (Invitrogen 3HH4-4G8), anti-H2Bub1 (Cell Signaling Technology, #5546), anti-H3K4me3 (Abcam ab8580), anti-H3K9ac (Merck-Millipore #07-352), Peroxidase AffiniPure F(ab') Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (Jackson ImmunoResearch #115-036-071) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch #115-035-144). Protein levels were quantified by ImageJ.
HCl was added to a final concentration of 0.2 M and incubated on an end-to-end rotator for 2 hours at 4°C. Following the incubation, cell extract was centrifuged at 20 800 x g for 10 min at 4°C, to pellet the acid insoluble material. A solution of 2 M Tris-HCl (pH 8.8) was added to neutralize the supernatant of the acidic extraction. Ten μl of the supernatant, containing histone proteins, were run on 4-12% gels (Bis-tris NuPAGE Novex, Life Technologies), then proteins were transferred and western blot assays were carried out by using standard methods. The following antibodies were used: anti-H3 (Abcam #ab1791) anti-H4 (Invitrogen 3HH4-4G8), anti-H2Bub1 (Cell Signaling Technology, #5546), anti-H3K4me3 (Abcam ab8580), anti-H3K9ac (Merck-Millipore #07-352), Peroxidase AffiniPure F(ab') Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (Jackson ImmunoResearch #115-036-071) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch #115-035-144). Protein levels were quantified by ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!