PROTEIN MEASUREMENT USING BICINCHONINIC ACID - ELIMINATION OF INTERFERING SUBSTANCES
PROTEIN MEASUREMENT USING BICINCHONINIC ACID - ELIMINATION OF INTERFERING SUBSTANCES
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DOI：
10.1016/0003-2697(89)90101-2
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发表时间：
1989-07-01
影响因子：
2.9
通讯作者：
HYLAND, KJ
中科院分区：
文献类型：
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作者：
BROWN, RE;JARVIS, KL;HYLAND, KJ
基于二辛可宁酸（BCA）的蛋白质定量方法简单、灵敏，并且对许多已知会干扰Lowry方法的去污剂和物质具有耐受性。然而，在蛋白质纯化过程中经常使用的某些化合物确实干扰BCA蛋白质测定。BCA发色团对各种干扰物质的响应提供了对BCA定量蛋白质的机制的深入了解。某些物质（例如，葡萄糖、巯基乙醇和二硫苏糖醇）与BCA工作试剂结合时，在562 nm处产生强吸光度。吸光度似乎与蛋白质引起的正常反应相同。其他代理（例如，硫酸铵和某些两性电解质）减少蛋白质诱导的显色并移动颜色响应的波长。这两种类型的干扰可以通过用脱氧胆酸盐和三氯乙酸选择性地沉淀蛋白质来消除（A。Bensadoun和D. 03 The Dog（1976）70，241-250），然后与二喹啉甲酸反应。本文所述的修饰允许快速、有效地去除蛋白质纯化过程中常用的许多干扰物质。
Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminsh the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70, 241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.