Lovrien Rex, Matulis Daumantas
University of Minnesota, St. Paul, Minnesota, USA.
Curr Protoc Microbiol. 2005 Oct;Appendix 3:Appendix 3A. doi: 10.1002/9780471729259.mca03as00.
This unit describes three copper-based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree-Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low-molecular-weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low-molecular-weight contaminants.
双缩脲法、Lowry法的一种变体（Hartree-Lowry法）和二辛可宁酸（BCA）测定法。蛋白质的酸水解与茚三酮检测相结合，以定量样品中的氨基酸含量。紫外分光光度法用于测量总蛋白并评估样品中是否存在污染物。考马斯亮蓝染料结合法（即Bradford法）是一种非常简单的测定方法，通常灵敏度较高，尽管有时根据蛋白质在酸性pH条件下与染料结合的好坏程度会给出可变的响应。最后，干重测量用于定量纯蛋白。辅助方案描述了用于酸水解的热封玻璃管、在聚丙烯酰胺凝胶孔中进行样品透析以去除低分子量污染物以及用三氯乙酸沉淀法沉淀和浓缩蛋白质并去除低分子量污染物。
