In vitro histone acetylation assays were performed on 25 μg of mitotic chromatin (isolated as described above and quantified using the bicinchoninic acid colorimetric assay system [Sigma-Aldrich]) in 50 mM Tris–HCL, 10% glycerol, 1 mM DTT, 1 mM PMSF, 50 nM TSA, 0.1 mM EDTA supplemented with 100 μM Acetyl-CoA, and 10 μg His-tagged histone H3 (#52023; BPS Bioscience). The reaction was incubated at 37°C for 1 h. HAT activity was quantified by: (i) a colorimetric assay quantifying the release of Co-A using DTNB (2-nitrobenzoic acid) (Sigma-Aldrich) by measuring the absorbance at 412 nm; (ii) isolating the His-tagged H3 by Ni-NTA column and measuring H3K9ac by immunoblots using anti-H3K9Ac antibody (Cell Signalling #C5B11; 1:1,000). All acetylation assays were performed in duplicate.
His tagged histone h3
Manufactured by BPS Biosciences
His-tagged histone H3 is a recombinant protein that contains a histidine (His) tag attached to the histone H3 protein. Histones are essential components of chromatin, the complex of DNA and proteins that make up the structure of chromosomes. This product can be used for various research applications that require purified histone H3 protein.
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Lab products found in correlation
2 protocols using his tagged histone h3
1
In Vitro Histone Acetylation Assay for Chromatin
2
Histone Acetylation Quantification Assay
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In vitro histone acetylation assays were performed on 25 μg of mitotic chromatin (isolated as described above and quantified using the bicinchoninic acid colorimetric assay system (Sigma)) in 50 mM Tris-HCL, 10% glycerol, 1mM DTT, 1mM PMSF, 50nM TSA, 0.1 mM EDTA supplemented with 100μM Acetyl-CoA and 10 μg Histagged Histone H3 (BPS Bioscience #52023). The reaction was incubated at 37°c for 1 h. HAT activity was quantified by: i) a colorimetric assay quantifying the release of Co-A using DTNB (2-nitrobenzoic acid) (Sigma) by measuring the absorbance at 412nm; ii) isolating the His-tagged H3 by Ni-NTA column and measuring H3K9ac by immunoblots using anti-H3K9Ac antibody (Cell Signalling # C5B11; 1:1000). All acetylation assays were performed in duplicate.
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