Concentrating protein lysates

Angela Alexander via methods%40net.bio.net (by udflutes from yahoo.com)
Mon Jul 2 06:27:59 EST 2007


I'm working with a new cell line that's giving me very dilute lysates (even with a vastly reduced volume of lysis buffer), such that I can barely load 15-20 micrograms of protein on a western gel. I'm doing a lot of signaling analysis and sometimes need more protein than that to get solid blots for some phospho-antibodies (we usually load 30-40). So I was wondering if you guys had any suggestions on the best method for concentrating my lysates without affecting my data ie losing robustness of phosphorylation. 
  
Speedvac'ing might be reasonable, as we have one in my lab right behind my bench, but I'm concerned the length of time the lysate will not be kept cold that will be required might not be such a great idea. Is dialysis the only way to go, or is there a quicker, easier method?....since I will be generating many long time courses/dose responses with these cells once I figure out the best method/something that works.

Thanks
Angela Alexander
Graduate Student
University of Texas MD Anderson Cancer Center

       
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