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在获得6个品种的玉米(Zea mays L.)花粉精细胞后,采用N-hydroxysuccinimido-biotin(NHS-biotin)标记其外膜蛋白,并通过SDS-PAGE和Western blotting对比了其中主要标记蛋白,发现其主要蛋白带差异并不显著,主要标记蛋白分子量均集中于91、60、43、30和17kD。采用免疫亲和层析技术进一步纯化已获得的混杂少量其它细胞器成分的精细胞质膜制剂,即利用制备的体细胞主要细胞器:线粒体、内质网、高尔基体及质膜的膜蛋白,分别免疫豚鼠,从其抗血清中纯化获得IgG,并进一步制成各种膜蛋白的免疫亲和吸附制剂。利用此技术进一步纯化经NHS-biotin标记的精细胞质膜蛋白,获得精细胞质膜特异的蛋白质,其中最为显著的蛋白质分子量约为65、22kD。
After obtained the pollen sperm cells of six maize varieties (Zea mays L.), the outer membrane proteins were labeled with N-hydroxysuccinimido-biotin (NHS-biotin), and the major marker proteins were compared by SDS-PAGE and Western blotting. Found that the main protein band difference is not significant, the main marker protein molecular weight are concentrated at 91,60,43,30 and 17kD. Immunocytochemistry was used to further purify the plasm membrane preparations that had been mixed with a few other organelles, namely, guinea pigs were immunized with the main organelles of somatic cells: mitochondria, endoplasmic reticulum, Golgi body and plasma membrane , IgG was purified from its antisera and further made into immunoaffinity adsorption preparations of various membrane proteins. Using this technology to further purify the NHS-biotin labeled sperm plasma membrane protein, to obtain sperm-specific plasma membrane protein, the most significant of which molecular weight of about 65,22 kD.