Im currently trying to see if the recombinant protein that Im expressing in Pichia is there, comparing uninduced vs induced media samples. Nevertheless I am not able to detect any secreted protein in the media by coomassie stained SDS-PAGE gels. Only by using silver stain I know I have protein in my samples, but I think I will need to concentrate (TCA precipitation) of higher volumes of supernatant to be able to see it by coomassie (3-6 ml). Any recommendation??
Hi
I think your question about protein concentration, for coomassie the minimum protein per well is 0.1 micro gram while for silver stain you need 2 ng in a single band and that's you have seen protein bands with silver stain but not with the former, you can work from that culture medium (how many mls used) that gave you this amount, I suggest to use several tubes or flasks but not one in this way you might be able to collect more protein (In very simple words; the yeast may release some enzyme in one flask which enough to degrade something in the medium but enough for you to see on SDS). Finally I would use ammonium sulphate for precipitation, its cheap and safe.
Hope this helps, Good luck
Hi, Ideally, you should be able to detect your protein without much hassle. After induction, I usually grow a one liter culture for around 20 hours. It gives me good concentration to be detected on a SDS-PAGE. After sonication, I pellet it down. The minimum amount that gives me a clear band is 10 microliters of supernatant + appropriate amount of loading dye. 30 microliters of supernatant + appropriate amount of loading dye gives me a very clear band.
But it all depends on the amount of protein expressed. The results may vary from protein to protein.
If you have further questions, please let me know.
Girish
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