蛋白含量检测的抗干扰新方法

New protein assay with improved tolerability to interferences

摘要:

为提高蛋白质含量检测方法的抗干扰能力，从福林酚试剂法入手，以牛血清白蛋白为标准样品，重新设计制定实验试剂组成与配比等，获得蛋白质含量检测新方法，然后探讨新方法的适用检测波长范围和稳定性，并利用细胞全蛋白裂解液分析它对多种常见干扰物质的包容性。实验发现，新方法不仅能准确检测蛋白质含量，对蛋白质测定液中表面活性剂的耐受浓度，如十二烷基硫酸钠 (SDS)、NP-40和TritonX-100分别达到10％、2％和1％；螯合剂乙二胺四乙酸二钠盐 (EDTA) 和Ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA) 则分别达25 mmol/L 和1 mmol/L；对还原剂二硫苏糖醇 (DTT) 和β-ME的包容浓度均为1 mmol/L；而含氮化合物硫酸铵与尿素则分别为0.5 mol/L和4 mol/L。与原方法相比较，对常见干扰物质的耐受性有显著提高，说明新方法适用含多种干扰物质的蛋白质溶液，在生命科学研究领域具有广泛应用前景。

Abstract:

Routine protein assays are usually affected with various compounds, and we need to use different protein quantification protocol to deal with different interference. In order to simplify the procedure, we developed a new method, in which the components and concentrations of the reagents were modified mainly based on classic Folin-Ciocalteu’s reagent for reducing the susceptibility to interfering substances. Standard curves of the new method were established with different levels of bovine serum albumin, and then, we assessed and evaluated the detectable wavelengths and stability. In particular, the tolerability to several interfering substances was analyzed by using cytolysis solutions containing different chemicals. Our data in this study show that the new method could be applied to detecting protein concentrations accurately, even in the presence of surfactants such as 10% sodium dodecyl sulfate (SDS), 2% NP-40, or 1% TrintonX-100, chelators of 25 mmol/L EDTA or 1 mmol/L Ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA), reductants of 1 mmol/L Dithiothretol (DTT) orβ-Mercaptoethanol (ME), or nitrogen-containing compounds of 0.5 mol/L ammonium sulphate or 4?mol/L urea. Taken together, these results indicate that the new approach significantly improves the tolerance to the interfering substances, which could be potentially useful in measuring the contents of proteins interfered with such substances.