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用5mol/L尿素,将大鼠Morris肝癌7777染色质解离为染色质非组蛋白 (UP组分)及染色质沉淀(UC组分)。UP(含90—95％非组蛋白)用免疫亲和层析(与大鼠Morris肝癌7777去组蛋白染色质抗体交联)分级,经2mol/L NaSCN及8mol/L尿素分部洗脱。将UP及UC,来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱组分同时进行SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)。以大鼠Morris肝癌7777去组蛋白染色质抗体作探针,进行免疫显迹(Immunoblot)测定。在UP部分出现二条阳性带,分子量为:200K及116K。UC部分有三条染色不很深的阳性带,分子量为200K,118K及91K。来自UP亲和层析的2mol/L NaSCN及8mol/L尿素洗脱部分分别有一条浓而清晰的阳性带,分子量分别为74K及83K。用酶联免疫吸附法(E1isa)测试从UP凝胶上切割下的阳性区带,其免疫特异性显著。
With 5mol / L urea, the rat Morris liver cancer 7777 chromatin was dissociated into chromatin non-histone (UP fraction) and chromatin precipitate (UC fraction). UP (90-95% non-histone) was fractionated by immunoaffinity chromatography (cross-linked with rat Morris hepatoma 7777 histone chromatin antibody) and eluted with 2 mol / L NaSCN and 8 mol / L urea. UP and UC, 2 mol / L NaSCN from UP affinity chromatography and 8 mol / L urea were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) simultaneously. The rat Morris hepatic carcinoma 7777 histone chromatin antibody was used as a probe to carry out immunoblot assay. In the UP section there are two positive bands, molecular weight: 200K and 116K. The UC section has three well-stained positive bands with molecular weights of 200K, 118K and 91K. The eluted fractions of 2mol / L NaSCN and 8mol / L urea from UP affinity chromatography had a strong and clear positive band with molecular weights of 74K and 83K, respectively. Positive bands cut from UP gels were tested by Elisa and the immunospecificity was significant.