Hi, I'm working on observing a protein on a western blot, I have been unsuccessful. I have tried concentrating my sample using acetone precipitation. I'm going to try and concentrate my sample more. any suggestion?
Also, I will be starting new cell cultures using complete media consisting of DMEM w/10% FBS. Is there anyway to help my cells produce more protein?
Re: Protein Quantitation.....
I guess if you can't even detect your protein via Western Blot, it must be in very very low concentration or it is even not there. When I grew cells on DMEM I measured the general concentration proteins in the cell lysate just per spectrophotometry at 600 nm (standardized by using BSA standard concentrations). For IP I used about 650 µg, for Western I always used about 30µg of the cell material and it worked perfectly fine.
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