Magnani M, Stocchi V, Serafini G, Chiarantini L
Istituto di Chimica Biologica, Università di Urbino.
Ital J Biochem. 1988 Mar-Apr;37(2):96-103.
Quantitation of stained, electroeluted proteins by the classical Lowry and Bradford protein assay is not possible because of some different interferences. In particular we have found that the substance interfering in the Lowry method cannot be removed by trichloroacetic acid precipitation nor can be compensated for by the appropriate blank. Interferences in the Bradford protein assay are due to detergents and pH of the protein buffer as well as to Coomassie brilliant blue R250 electroeluted with the protein sample. However, while these interferences can be compensated for by appropriate blank and standard curves, others (probably due to acrylamide fines) cannot be corrected. All these problems can be overcome by concentration and dialysis of electroeluted samples which permit the removal of interfering substances and the use of Bradford and Lowry protein assay in the 1-20 micrograms range, respectively. Successful applications are described for electroeluted bovine serum albumin, human hexokinase and phosphoglucomutase.
由于存在一些不同的干扰因素，无法通过经典的洛瑞法和布拉德福德蛋白测定法对染色、电洗脱后的蛋白质进行定量。特别是我们发现，干扰洛瑞法的物质不能通过三氯乙酸沉淀去除，也不能通过适当的空白对照来补偿。布拉德福德蛋白测定法中的干扰因素包括洗涤剂、蛋白质缓冲液的pH值以及与蛋白质样品一起电洗脱的考马斯亮蓝R250。然而，虽然这些干扰可以通过适当的空白对照和标准曲线来补偿，但其他干扰（可能是由于丙烯酰胺细粉）无法校正。通过对电洗脱样品进行浓缩和透析可以克服所有这些问题，这允许去除干扰物质，并分别在1 - 20微克范围内使用布拉德福德和洛瑞蛋白测定法。文中描述了电洗脱牛血清白蛋白、人己糖激酶和磷酸葡萄糖变位酶的成功应用。
