I'm planning to assay arginase1 activity on peritoneal macrophages, but in the litterature I found that some authors add protease inhibitor in the lysis buffer and some don't. I'd like to know if i can do this assay without proteases and expect some good results
When lysing cells, it is always possible for the proteins to be degraded by proteases unless you add inhibitors. You can try it without adding inhibitors, but the quality of your results will depend on the time after you lyse the cells and the way that you store the lysates, etc. The sooner you do the assay the better, unless you are purifying the protein from the lysate. Some proteins degrade much more rapidly than others. I don't know how susceptible arginase 1 is to degradation. My personal preference is always to add inhibitors, at least for the first try. You can always try after without the inhibitors and see how much difference it makes and remove it from the protocol if it has negligible effect.
Hi Anissa, I agree with Karen in that it is always good to have proteinase inhibitors present when dealing with tissue or cell lysates to prevent lysis of your protein.
Just be sure that the inhibitors do not affect your enzyme..
Take small portion of extract without protease inhibitors and assay for activity once in a week and check for stability over a period of time. If you don't loose activity due degradation of arginase by protease, don't add inhibitors otherwise you should add protease inhibitors to protect your enzyme from protease
Hi Anisa, It is always good to have sufficient proteinase inhibitor present when you deal with protein assay.
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