Venous blood was collected with citrate anticoagulant and immediately centrifuged at 1500 × g for 25 min at room temperature. Plasma was collected and alanine aminotransferase (ALT) levels were measured by an automatic blood biochemical analyzer in the clinical laboratory and histone levels were measured by western blotting using anti-histone H3 (Abcam), as described previously[32 (link)]. Plasma and histone H3 protein (New England Biolabs, Herts, United Kingdom) as standard were subjected to SDS-PAGE and electrically transferred onto PVDF membranes (Millipore). After blocking, the blot was probed by anti-histone H3 (Abcam, 1:2000) at 4 °C overnight. After extensive washings, HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:10000) was incubated at room temperature for 1 h. After extensive washing in TBST, ECL (Millipore) was used to visualize the protein bands, which were quantified against histone H3 protein standard to obtain the concentration of histone H3 in plasma using Image J software. The total histones were estimated based on the molar ratio of H3 in the cell nuclei.
Histone h3 protein
Manufactured by Merck Group
Sourced in United States
Histone H3 protein is a core component of nucleosomes, the fundamental units of chromatin in eukaryotic cells. It plays a crucial role in the organization and compaction of DNA within the cell nucleus. Histone H3 is essential for the regulation of gene expression and other cellular processes that involve chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Histone h3, by Merck Group (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Cell Signaling Technology (1 mentions)
Phospho histone h3 antibody, by Cell Signaling Technology (1 mentions)
Dialysis tubing, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
9 10 phenanthrenequinone, by Merck Group (1 mentions)
3 deoxyglucosone 3 dg, by Merck Group (1 mentions)
4 protocols using histone h3 protein
1
Plasma Histone H3 Quantification
2
Kinase Activity Assay for CDK1 and Aurora-B
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To examine cdk1 and Aurora-B kinase activity, respective primary antibodies were used to selectively immunoprecipitate protein-containing complexes from cell lysates. The resulting immunoprecipitate was incubated with pure Histone-H3 protein in the presence of 32P-labelled ATP and kinase buffer. The kinase assay reaction allowed immunoprecipitated cdk1 to phosphorylate histone-H3 in vitro, the extent of which was measured by immunoblotting using phosphohistone-H3 antibody from Cell Signaling (MA, USA). Histone-H3 protein was from Millipore (MA, USA) and ATP was from Cell Signaling.
3
Quantifying Histone Modifications by 3-DG
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Histone protein H3, 3-deoxyglucosone (3-DG), arginine, lysine, sodium azide, 2,4-dinitrophenyl hydrazine (DNPH), 9,10-phenanthrenequinone, dialysis tubing and other reagents/chemicals were acquired from Sigma Chemical Company, USA. NBT was purchased from Sisco Research Laboratories, India.
4
Purification and Characterization of Histone Proteins
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All chemicals were purchased from ACROS Organics or Sigma Aldrich and used without further purification. Solvents were of chemical grade and were used without any purification. Water was obtained on a System Direct-Q 3 Reverse Osmosis Purifier (Merck, Japan) and used with a resistivity of 18 MOhm. All histone protein types and nucleosome have been purified from calf thymus and supplied in lyophilized powder. Histone protein H2A (reference H9250), Histone protein H3 (reference 11034758001) and nucleosome containing a mixture of histone proteins H1, H2A, H2B, H3, and H4 (reference 10223565001) have been purchased from Sigma Aldrich. The histone protein duplex H3/H4 (Reference MBS634742) has been purchased from the MyBioSource company. Lyophilized histone proteins and nucleosome samples were re-suspended in aqueous solution. The pH of sample solution was measured using a compact pH meter, twin pH (Horiba, Japan). A table reporting the pH values is given in the Supplementary Data (Supplementary Table S1 ).
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