First I used only PMSF, and now I am trying the cOmplete cocktail tablets. I need to incubate protein extracts and my protein of interest is degraded and I cannot analyze the experiment. Any suggestion for stronger proteases inhibitors? My next step will be to try to heat the sample, but I am afraid that my protein will also be degraded.
Thanks!
Hi Crisitna
I used the complete cocktail and wirked...but in case it doesnt here is a paper describing different protease inhibitors (in Table 5.25.2).
http://arrow.dit.ie/cgi/viewcontent.cgi?article=1004&context=ehsiart
It was really useful for me when I needed alternatives
I hope it helps you
Regards
Marco
Hi Marco!
Thank you so much for you answer! Thank you so much for your reference!
Did you incubate your protein extracts 4 days at 37ºC with cOmplete and you didn't had protein degradation? Were you adding cOmplete every day? at 1x or higher concentrations? My idea is not to purify the proteins and to work with the protein extract.
Thanks again!
Cristina
I dont remember using it at 37C, I think we use them at room temperature and the samples were later stable in teh fridge for longer time. The proteins that we were studying were vegetable proteins and maybe it could be some differences in stability if you are isolating animal proteins.
In any case you could see if there's protein degradation or not after using it by taking small samples (10 uL) and doing a simple SDS gel...to be fully sure of your degradation profile.
Depending also on the use you want to give to your proteins you could also consider freezing them or freeze-drying them. It gives much better stability, but again it depends on the future applications you have in mind
Good luck
Marco
Hi Cristina,
Incubating at 37C for four days will most certainly lead to degradation of almost all proteins to various degrees. We at Denator have worked a lot with preventing protein degradation using heat denaturing of enzymes using the Stabilizor system. Heat denaturation of enzymes is generally an efficient way of stopping enzymatic activity and could potentially also work for you research question as long as you are not depending on some other enzymatic activity in your sample. Checking the progression of degradation, as suggested by Marco, is a very good way to understand the problem and the time variable. Check out our webpage, www.denator.com for more information and contact info for further discussion on your particular problem.
Best regards,
Mats
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