I am trying to do an 2-DE after ACN precipitation but when I do the IEF, I do not enough reach the voltage that I need (for example, in the first step, I need 250V but I only have 15V and the amperage is too high), which could be the problem?
Hi Juan,
it could be that you have too much salt in your samples. Have you cleaned your pellet thoroughly before you resolved your sample with the IEF compatible puffer? What are the ingredients of your used IEF buffer? With 15V is definitely something severly wrong.
Kristin
Thank you for the answer, I am using a standard rehidratation buffer and I used it for another IEFs with good results so I think this is not the problem. Probably we have an excess of salt, I will do a clean up before the IEF. Thank you for your help!
I agree with Kristin. Your sample seems to have a lot of salt. Try desalting columns and give buffer exchange with 1mM Tris, pH 8.0 along with appropriate protease inhibitors. Finally concentrate the sample to 10 ug/ul. Resuspend in rehydration buffer. I am sure this should work. All the best.
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