Top quality substrates for protein kinases, methylases and deacetylases
Yorkshire Bioscience offers high-quality histones isolated from calf thymus for use as substrates in protein kinase and methylase reactions.
Histones are basic proteins that are found complexed with DNA in cell nuclei of eukaryotic cells. Bacteria do not have histones. However, the DNA of the genus Thermoplasma (in the domain Archaea) is surrounded by a highly basic DNA-binding protein which strongly resembles the eukaryotic histones.
A chromosome contains five types of histones: H1 (or H5), H2A, H2B, H3 and H4. H1 and its homologous protein H5 are involved in higher-order structures. The other four types of histones associate with DNA to form nucleosomes. H1 (or H5) has about 220 residues. The other types of histones are smaller, each consisting of 100-150 residues.
An important feature about histones is that they contain a few lysine (K) residues at the N terminus. Under normal cellular conditions, the R group of lysine is positively charged, which allows it to interact with the negatively charged phosphates in DNA. The positive R group of lysine may be neutralized by acetylation, reducing the binding force between histones and DNA. This mechanism has been demonstrated to play a major role in the regulation of gene transcription.
Article for review: Origin of H1 linker histones - FASEB J., 2001.
All histones are also available in bulk quantities up to 100 g of each.
Shipment: ambient temperature.
Store in dry dark place at -20 ºC for more than 24 months.
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In Vitro Assay of Protein Kinase Activity -- Samples of the recombinant kinases were incubated in phosphorylation buffer (25 mM Hepes, 5 mM MgCl2, 5 mM MnCl2, 0.5 mM dithiothreitol) containing 10 µM [-32P]ATP (100 µCi/ml) for 10 min or 30 min for different preparations at room temperature. Substrates (histones) were added to a final concentration of 0.1 - 0.25 mg/ml. Reaction products were separated by SDS-PAGE (16% gels) and visualized by autoradiography of the dried gels.
In Vitro Methyltransferase Assays -- 50 µl of reaction mixture containing substrates (a mixture of histones H1, 2A, 2B, 3, and 4; and single H1, H2A, and H3), enzymes, and 250 nCi of S-adenosyl-[methyl-14C]-L-methionine in methylase activity buffer (50 mM Tris, pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM -mercaptoethanol, 250 mM sucrose) was incubated for 60 min at 37 °C. The reaction products were separated by 15% SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Brilliant Blue R-250 staining and by autoradiography of the dried gels.