The Identification Of Histones Eliminylation Sites

In recent years, researchers work to finding more eliminylated modified polypeptide and protein in order to explain life activities and fill in this blank field. But there are no better research methods. Histones eliminylation modification sites identified as well.Unsaturated amino acid can attack michaelcovalent addition through the thiol group. For identification of the eliminylated modified protein and polypeptide,we can chemically label them through sulfhydryl reagent. and ultimately affinity enrichment to get specific eliminylated protein or polypeptide.Analyzing five kinds of amino acid composition of histones containing H1, H2A, H2B, H3, and H4, we found that five kinds of histones have a high proportion of eliminylation amino acid, including serine and threonine. We suppose that histones are likely to contain eliminylation sites. In addition to the H3 contains one cysteine, the other four histones have no cysteine. Because of the particularity of histones amino acid composition, the experiment method is feasible for us to provide the reliable basis, also provides convenient for our later specificity of thiol purification process to remove. To identify histone eliminylation sites, we can choose dithiothreitol (DTT),which is a small molecules containing double thiol reagents. Among them, one thiol group open eliminylated unsaturated amino acids in order to label histones DTT-tag; another expose of sulfhydryl group make us purify histones by thiol-activated agarose affinity column. Finally, we can attain specific labelled histones.Firstly, we selected cattle brain tissue, washed nuclei repeatedly, extrated histones by acid and salt. Using acid extraction of histones optimize the best conditions of addition reaction.The detergent properties and small molecule removal conditions required for comparison, then selecting the glycerol which is small molecular weight, easy to remove the detergent through the dialysis . Then, we found that the optimal reaction concentration of glycerol in the reaction was 8%. Then compared to several general buffer, the results show that several buffer has a relatively strong responsive signal, among them sodium bicarbonate is the strongest. At last, the reaction signal of temperature is compared and the results show that the room temperature is the optimum reaction temperature.Then, the optimal purification conditions of the affinity enriched group proteins were optimized..The results of the comparison of the column efficiency of the labeled groups at different temperatures show that the room temperature has a strong incubation efficiency. Comparing to affinity enrichment of chemically labelled histones using different buffer, the results showed that histone in Tris and NaHCO3 have strong incubation efficiency, HEPES and NaHCO3 can be purified to high specificity of histones. Finally, when the purified histones was precleaned and the high concentration urea was selected to remove the non-specific binding protein,we got the specific binding of the histone.This paper is established in our laboratory mature labeling probe. Next, we detected and optimized reaction conditions respectively from the buffer, detergent, reaction temperature and time. The best reaction conditions of chemical markers has been selected, followed by the cysteine standard curve to detect the content of free sulfhydryl group of thiol labeled samples, followed by sulfhydryl activated agarose column specificity to purify target protein. This study provides a theoretical basis for the further study of the identification of eliminylated protein and polypeptide.