毛白杨形成层总蛋白裂解及蛋白含量测定方法优化

Optimization of protein lysis and determination of protein content for Populus tomentosa cambium tissues

通过TCA/丙酮法研磨提取获得毛白杨形成层总蛋白质粉末。在室温和冰浴条件下,用不同的裂解液对粗提总蛋白进行裂解,分别在1,2及3h取出裂解蛋白液。通过聚丙烯酰胺凝胶电泳和双向电泳对蛋白质裂解情况进行检测、比较与分析,获得了蛋白质最适裂解条件为:蛋白裂解液(8mol/L尿素,2mol/L硫脲,4%CHAPS,65mmol/L DTT,2%Bio-lyte)在室温条件裂解3h。同时对蛋白质含量测定方法-Bradford法及2种改良的Bradford方法进行了比较,最终获得了用裂解缓冲液配制蛋白质标准曲线系列溶液以降低本底干扰的优化蛋白质含量测定方法。

TCA/acetone was used to extract total crude protein from cambium tissues of Populus tomentosa. Six kinds of protein lysis buffers were used to dissolve and lysis the total protein under room temperature and ice-bath, respectively. Lysis processes were monitored by sampling at the interval of 1, 2and 3 h. The samples were determined and analyzed with SDS-polyacrylamide gel electrophoresis and the 2-DE gels electrophoresis. Optimal lysis buffer were composed of 8 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 65 mmol/L DTT and 2% Bio-lyte and improved lysis condition were found to be at room temperature for 3 h. Comparison of Bradford method with two other improved methods was done for protein content determination and the best one is to reduce the background interference by the preparation of BSA standard curve solution with the lysis buffer.