Ribosome samples were separated on NuPage 4–12% gradient Bis-Tris gels (Invitrogen), and transferred to a nitrocellulose membrane (Bio-Rad) by wet blotting. Fluorescently labeled RPL10 was probed by monoclonal mouse anti-RPL10 antibody (Thermo Fisher, MA526901, 1:2000). H2B were probed by polyclonal anti-H2B antibody (Abcam, ab1790). Horse radish peroxidase anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, 1:5000) were visualized using ECL Plus Western Blotting Substrate (Thermo Scientific) on a Bio-Rad ChemiDoc Imager. BAF-depleted samples were immunoblotted as previously described15 (link).
Anti h2b antibody
Manufactured by Abcam
Sourced in United Kingdom
The Anti-H2B antibody is a specific antibody that recognizes the histone protein H2B. Histones are core components of chromatin, the complex of DNA and proteins that make up the structure of chromosomes. This antibody can be used to detect and study the H2B histone protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Chemidoc imager, by Bio-Rad (2 mentions)
Monoclonal mouse anti rpl10 antibody, by Thermo Fisher Scientific (2 mentions)
Horse radish peroxidase anti mouse or anti rabbit secondary antibodies, by Bio-Rad (2 mentions)
Ipfl00010, by Merck Group (2 mentions)
Imagej, by Bio-Rad (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Anti h3k27ac antibody, by Active Motif (2 mentions)
Anti h3k9ac antibody, by Active Motif (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Ecl detection kit, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using anti h2b antibody
1
Ribosome Profiling via Western Blot
2
SIRT6 Deacetylation Kinetics on Nucleosomes
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Wild-type SIRT6 (500nM or 125nM) and SIRT6(R175A) (200nM) was incubated with nucleosomes (125nM) containing either acetylated H3K9, H3K56, or H3K27, and/or with truncated H2A (H2At) at 37 °C for varying time points in HDAC reaction buffer (25mM HEPES pH 7.3, 49 mM NaCl, 4.5mM MgCl2, 1mM DTT). Reactions were quenched with the addition of 2X SDS sample buffer, boiled, and then electrophoresed on a 12 % SDS-PAGE gel. Proteins on the gel were transferred to a PVDF membrane (Millipore IPFL 00010), which was first blocked with 10 % milk, and then probed with either 1:10,000 anti-H3K9ac antibody (Active Motif #39038), 1:1,000 anti-H3K56ac antibody (Active Motif #39133), or 1:3,000 anti-H3K27ac antibody (Active Motif #39082), and also probed with 1:3,000 anti-H2B antibody (Abcam #64039). Western blots were quantified using ImageJ (54 (link)) or Bio-Rad Image Lab.
3
Ribosome Profiling via Western Blot
4
SIRT6 Deacetylation of Histone Modifications
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Wild-type SIRT6 (500 or 125 nM) and SIRT6(R175A) (200 nM) were incubated with nucleosomes (125 nM) containing either acetylated H3 K9, H3 K56, or H3 K27, and/or with C-terminally truncated H2A [H2A(2-119)] at 37°C for varying time points in HDAC reaction buffer [25 mM Hepes (pH 7.3), 49 mM NaCl, 4.5 mM MgCl2, and 1 mM DTT]. Reactions were quenched with the addition of 2× SDS sample buffer, boiled, and then electrophoresed on a 12% SDS-PAGE gel. Proteins on the gel were transferred to a polyvinylidene difluoride membrane (Millipore, IPFL 00010), which was first blocked with 10% milk, and then probed with either 1:10,000 anti-H3K9ac antibody (Active Motif, #39038), 1:1000 anti-H3K56ac antibody (Active Motif, #39133), or 1:3000 anti-H3K27ac antibody (Active Motif, #39082), and also probed with 1:3000 anti-H2B antibody (Abcam, #64039). Western blots were quantified using ImageJ (54 (link)) or Bio-Rad Image Lab. Control experiments show similar SIRT6 deacetylase activity using nucleosomes reconstituted from H3K9ac protein prepared in-house or purchased from the Histone Source.
5
Western Blot Analysis of Protein Expression
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Treated CHSE/F cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich, San Luis, Missouri, MO, USA). Proteins were denatured at 95 °C for 5 min, separated by SDS-polyacrylamide gel electrophoresis (10–12%), and then were transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). The membrane was incubated with PBS 1× containing 5% dehydrated skim milk at 4 °C. The membrane was incubated with anti-mCherry (1:2000, Abexxa, Cambridge, UK), anti-Flag antibody (1:2000, Millipore), anti-H2B antibody (1:2000, Abcam, Cambridge, UK), followed by horseradish peroxidase conjugated goat anti-mouse IgG antibody (1:5000, Invitrogen, Carlsbad, CA, USA). Bands on X-O-mat Blue films (AGFA) were visualized via enhanced chemiluminescence (ECL detection kit; Amersham, Little Chalfont, UK) according to the manufacturer’s instructions.
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