Concentrating protein lysates

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Mon Jul 2 16:43:32 EST 2007


On 7/2/07, Angela Alexander <udflutes from yahoo.com> wrote:
> 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.


You did'nt mention what the starting volume is. I can think of these
small tubes with a membrane, which would concentrate the volume when
spun down. Frankly, I don't remember the company or catalog, however,
I imagine they should'nt be difficult to find.

If your starting volumes are rather large, like 2-10 ml, you could use
ultrafiltration methods.
>


best,
pow
> 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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