情况综述

产品

Roche罗氏公司蛋白酶抑制剂Cocktail片剂：cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets provided in EASYpacks

优点

使用方便，高效的抑制蛋白酶活性的混合片剂

缺点

混合片剂并不是抑制所有的蛋白酶活性，因此在您的实验过程中，针对某种特殊的蛋白酶，可能还需要再加入一两种特异的蛋白酶抑制剂

总结

作为常规实验室有关蛋白的操作，这种蛋白酶抑制剂预混药片是非常有用的。但对于某些比较独特的蛋白研究实验室，则需要根据需要配置自己需要的蛋白酶抑制剂混合物

One major barrier to protein research is the instability of protein. DNA is stable, and better yet DNAse is extremely unstable. RNA is stable, but RNAse is everywhere and extremely stable. Protein is unstable, and there are thousands of different proteases ready to cleave your protein of interest down to a couple dozen amino acids. The worst part is that these proteases are stable and active at various temperatures and conditions (depending on the protease).

To work with protein, protease inhibitors are essential. Once a cell is lysed, proteases are released from their restricted organelles. In order to collect that protein, and perform experiments, proteases must be inhibited to ensure the protein of interest is stable. Without extensive analysis of your protein solution, the proper cocktail of protease inhibitors is not easily generated. The most common proteases are cysteine and serine proteases in animal, plant, and bacterial cells. Roche has provided a simple and effective alternative to adding half a dozen individual protease inhibitors to effectively inhibit cysteines and serine proteases. Once the experiment-specific solution is made, just add 1 tablet per 10 mls (for 50 mls of solution there is a Complete Protease Tablet). After the tablet is dissolved, you have one week at 4°C before the inhibitors are no longer effective, or three months if frozen at – 20°C.

I have used these tablets extensively in my research. Although a lot of protein work involves SDS-PAGE and Western blotting techniques that do not require protease inhibitors, an increasing list of procedures require the use of protease inhibitors, including immunoprecipitation, protein fractionation, and electromobility shift assay (EMSA) to name a few. I have specifically used these tablets with immunoprecipitations, and have found these inhibitors to be a requirement for reproducible pulldowns. Without these inhibitors, I see several proteins that have been cleaved, and some proteins that are no longer efficiently pulled down, due in part to protease cleavage of these proteins.

One problem arises with these inhibitors. They do not inhibit all known proteases, including aspartic proteases and most metalloproteases. Luckily, aspartic proteases are active solely at acidic pHs, so a buffered solution will efficiently inhibit these proteases. However, if your lab works with metalloproteases, obviously this product is not for you.

Jeffrey Perry

Lab Manager
Microbiology and Immunology
University of Michigan
United States

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