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Are protease inhibitors overrated ?


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#1 Devil Fish

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Posted 30 December 2010 - 11:50 AM

I am a newbie to protein extraction world. One of my colleagues told me to get some Protease Inhibitor Cocktail & other one said not to bother much or make one using a homemade recipe. Luckily, I got a sample of Proteoguard Protease Inhibitor cocktail from Clontech with which I am quite satisfied. My proteins didnít degrade & the isolation was just what I wanted. But I am debating that for the next ones should I buy this mix or just not worry about it. ? What have been your experiences between a home made recipe & a commercially purchased product? Do you always use them? We are a very small lab working on a budget. Any thoughts would be appreciated.

#2 mdfenko

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Posted 30 December 2010 - 12:08 PM

as someone who has worked for years with various proteases, i completely endorse the use of inhibitors when isolating non-protease proteins.

some proteases work very quickly and will maintain some activity at low temperatures. some, what you may consider, minor handling differences may lead to large differences in yield and condition of your protein of interest.

the best commercial inhibitor cocktails cover all known classes of protease.

if you want to make your own cocktail then you will have to do the same. keep in mind that some inhibitors require careful handling and some are relatively labile.
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#3 SonjaK

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Posted 30 December 2010 - 01:27 PM

I have worked several years with protein purification and have found the use of protease inhibitors very helpful. If your protein of interest will survive without the use of protease inhibitor is impossible to guess, but if you have a good purification protocol and your purification is quick you may manage without an inhibitor. In that case try to keep your protein mix really cool (all reactions in the mix will be slow) and do the purification steps as quick as possible (especially the first ones when the mix is still really dirty).

Keep in mind that if your protein is easily degraded, the price of a protease inhibitor in quickly paid back by a better yield of the protein. Hope you find a good protocol!




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