摘要
本文描述了作者在研究低等生物着丝粒蛋白过程中对免疫印迹技术所作的一些改进：１．分离胶由１２％ＳＤＳ聚丙烯酰胺均一胶改作５％—１２％（或１５％）的线性梯度胶，使不同分子量范围的蛋白都能得以较好地分离；２．用眼虫色素代替溴酚蓝作示踪剂，以能适时地终止电泳；３．转移由４００ｍＡ３．５ｈ改为２００ｍＡ１７ｈ，由冰箱内空气冷却改为流水浴冷却，提高了冷却效率；４．对转移后的硝酸纤维素膜作除ＳＤＳ处理；使转移后的蛋白在一定程度上得以复性；５．抗体孵育温度由３７℃或室温１—２ｈ改为４℃冰箱过夜，降低了本底，提高了印迹反应的特异性。我们已用改进后的方法对一些低等生物作了一系列的着丝粒蛋白研究，取得了比较满意的结果。
The authors made some improvments of the protein immunoblotting techniques during studies on the centromere proteins in sone protists. The main modifications reported in this paper are as followings.1. Separating gels were modified from a uniform polyacrylamide gel of 12% to linear gradient gels of 5%-12%(or 15%), stacking gel from 5% to 3%, and the separation of pro tein bands in the gel was much improved. 2. Traching dye was changed from bromphenol blue to theplgments in Euglenagracilis SDS-PAGE sample,and the proper electrophoresis stop can be conveniently determined. 3. Electrophoretic transfer was changed from 400 mA, 3. 5 hurs to 200 mA. 17 hurs; the cooling of transfer from air in refrigerator of 4 degree centigrades to flowing water bath, so that the transfer was cooled more successfully. 4. The NC memberanes were washed thoroughly with double distilled water, then treat edwithwith0.1% TritonX-100-TBS aftertransfer, in orderto remove SDS in the protein bands transfered. 5. The incubation of antigen-antibody reactions were modified from RT or 37 degree centigrades, 1-2 hurs to 4 degree centigrades, thus the backgroung of blotting was much reduced and the specificity of antigen-antibody reactions were much improved.
关键词
免疫印迹
电泳
生化技术
Western immunoblotting, Electorphoresis, Electrophoretic transfer
