适于SDS-PAGE分析的高粱叶片蛋白质提取方法

摘要

为了探索适于高粱叶片蛋白质SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析的样品制备方法,对TCA/丙(酮)沉淀后的蛋白质分别用裂解液Ⅰ(尿素+硫脲+CHAPS+DTT)和裂解液Ⅱ(尿素+ DTT)2种裂解液进行裂解,用改良的Bradford法进行定量分析,并进行SDS-PAGE分离检测.结果表明,(裂)解液Ⅰ所得蛋白质质量溶度(2.538 g/L)高于裂解液Ⅱ所得蛋白质质量浓度(1.905 g/L),且两者呈极显著差异(P＜0.05),但裂解液Ⅰ所得蛋白质电泳(条)带出现1条杂带；而裂解液Ⅱ所得蛋白质能满足上样要求且电泳条带清晰无杂带.因此,TCA/丙酮沉淀并用裂解液Ⅱ进行裂解的方法适用于高粱叶片的SDS-PAGE分析.%In order to develop a sample preparation method suitable for SDS-PAGE analysis of sorghum leaf protein,the protein (after TCA/aceton precipitation) was resolubilized with lysis buffer Ⅰ (urea,thiourea,CHAPS and DTT) and lysis buffer Ⅱ (urea,DTT),respectively.And then the protein was quantified with modified Bradford assay,and was seperated by SDS-PAGE.The results showed that the concentration of protein(2.538 g/L) got from lysis buffer Ⅰ was higher than that from buffer Ⅱ (1.905 g/L),the difference being significant (P＜0.05).But an unexpected band of protein electrophoresis was appeared from lysis buffer Ⅰ ;whereas,enough protein was got from lysis buffer Ⅱ,and the bands were sharp and clear with no extra band.So the method of resolubilizing TCA/aceton precipitated protein with lysis buffer Ⅱ was suitable for SDS-PAGE analysis of sorghum leaf.