Simpson Richard J
CSH Protoc. 2008 Aug 1;2008:pdb.prot4722. doi: 10.1101/pdb.prot4722.
INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu(+) generated under alkaline conditions at sites of complexes between Cu(2+) and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.
引言
本方案描述了一种蛋白质定量方法，它是洛瑞测定法的一种变体。它使用二辛可宁酸（BCA）来增强在碱性条件下Cu(2+)与蛋白质形成的复合物位点处生成的Cu(+)的检测。生成的发色团在562nm处有吸收。该技术分为三个部分：标准程序、微量程序和96孔微量滴定板程序。对于每个程序，测试样品与用于生成校准曲线的蛋白质标准品平行测定，然后内插得出测试样品中蛋白质的准确浓度。标准BCA测定法使用大量的试剂和样品，且不易实现自动化。如果这些问题很重要，建议采用微量程序。进而它可以适用于96孔微量滴定板程序中的酶标仪。如果酶标仪与计算机连接，每小时可读取1000多个样品。
