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

作　　者：谢进[1] 侯佳音[1] 刘淑欣[1] 蒋湘宁[1] 盖颖[1] 

机构地区：[1]北京林业大学生物化学与技术学院,北京100083

出　　处：《河北农业大学学报》2013年第5期60-64,共5页Journal of Hebei Agricultural University

基　　金：国家高技术研究发展计划(2011aa100203);国家自然科学基金重点项目(30630053)

摘　　要：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.

关 键 词：毛白杨形成层 蛋白裂解 含量测定 优化 双向电泳 

分 类 号：S792.117[农业科学—林木遗传育种]