Assay procedure: the histones obtained as was described before were diluted to 300 ng/uL and were used as the protocol described.
With a starting material of 2♱0^8 cel/mL we obtained aproximately 6000 ug/mL of histones. After this incubation step, we proceed with a common precipitation with TCA/Acetone and the final protein pellet was solubilized in molecular biology water. For that reason, we chose another methology (used routinely in our lab): dissociated tissue was subjected to hypotonic lysis buffer (10mM Tris-Cl pH 8.0 1mM KCl 1.5 mM MgCl2 1mM DTT) and the obtained pellet was solubilized in HCl 0.4 N (to obtain the basic proteins). When the tissue was resuspended appeared a mucus material that made impossible to resuspend well the tissue. The difficult was to resuspended the tissue (or cells when dissociated) in the Lysis Buffer. Extraction of histones: the protocol described in the datasheed did not work well. Sample Type: Cells dissociated from zebrafish testicle. The kit is suitable for specifically measuring global histone H3 acetylation using a variety of mammalian cells including fresh and frozen tissues, and cultured adherent and suspension cells. The kit is ready-to-use and provides all the essential components needed to carry out a successful assay experiment. Histone H3 Acetylation Assay Kit (ab115102) allows the user to measure global acetylation of histone H3 at tremendously fast speeds and consistency, superior and safer than all other current methods.
An imbalance in the equilibrium of histone acetylation has been associated with tumorigenesis and cancer progression. Histone acetylation is tightly involved in cell cycle regulation, cell proliferation and apoptosis. HATs (histone acetyltransferases) and HDACs (histone deacetylases) play a critical role in controlling histone H3 actylation. However, unlike the latter, the H3 receptor is down-regulated by the divalent cation.Acetylation of histones such histone H3 has been involved in the regulation of chromatin structure and the recruitment of transcription factors to gene promoters. It is concluded that the H3 receptor, like many other amine receptors, is coupled to its still unidentified effector system via a G-protein and regulated by Ca2+. The presence of guanylnucleotides in the Krebs-Ringer medium with Ca2+ abolished the binding to this low-affinity component whereas in a phosphate buffer only the KD was slightly increased.
The presence of 2.6 mM Ca2+, in a modified Krebs-Ringer medium, promoted the conversion of a larger fraction of sites into a low-affinity component with a KD of 16 nM. Among antagonists burimamide was the only one to compete with a pseudo-Hill coefficient significantly different from unity (nH = 0.48 +/- 0.03), indicating a possible heterogeneity among binding sites. However, whereas the potency of antagonists was closely similar in the two assay systems, that of agonists was approximately 10-fold higher in the binding assay. Competition studies indicated that the binding occurred with a stereoselectivity and pharmacological specificity similar to that of functional H3 autoreceptors regulating histamine release in brain slices. The KD, derived from either dissociation/association rates or saturation kinetics at equilibrium, was approximately 0.5 nM at 25 degrees C. In phosphate buffer the specific binding defined with thioperamide, an H3 receptor antagonist, displayed characters of reversibility and saturability with a Bmax of approximately 30 fmol/mg protein. The binding of (R)alpha-methylhistamine, a potent and specific agonist at histamine H3 receptors, was investigated with membranes of rat cerebral cortex.