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Sample (protein) recovery on 2D gel - (Jun/12/2008 )

Hi all,
I had a fraction from ion exchange chromatography containing my protein (a cytosolic protein). 10% of the fraction I precipited by acetone and analysed by normal western blot (using normal SDS PAGE). I could detect clear, strong signal of my protein. Then I precipited the rest of sample (90%) again by acetone and I let analysed on 2D gel. The 2D gel was done at a department of proteomic - I did not do it by myself. I did only incubation with antibodies and detection of proteins on PVDF membrane from 2D. The protein signal on the PVDF membrane from 2D was very weak in spite of the fact, that it was used much higher amount of sample (90% of sample) than for 1D gel (10% of sample).
My question for those who have experince in 2D gels:
Is it normal, that at the 2D gel is so bad protein recovery (in comparison to 1D) or is not normal and there was problem, for example, at transfer on PVDF, solubilization of acetone precipitate etc.?

Thanks.
vic

-victor.m-

QUOTE (victor.m @ Jun 12 2008, 09:09 AM)
Hi all,
I had a fraction from ion exchange chromatography containing my protein (a cytosolic protein). 10% of the fraction I precipited by acetone and analysed by normal western blot (using normal SDS PAGE). I could detect clear, strong signal of my protein. Then I precipited the rest of sample (90%) again by acetone and I let analysed on 2D gel. The 2D gel was done at a department of proteomic - I did not do it by myself. I did only incubation with antibodies and detection of proteins on PVDF membrane from 2D. The protein signal on the PVDF membrane from 2D was very weak in spite of the fact, that it was used much higher amount of sample (90% of sample) than for 1D gel (10% of sample).
My question for those who have experince in 2D gels:
Is it normal, that at the 2D gel is so bad protein recovery (in comparison to 1D) or is not normal and there was problem, for example, at transfer on PVDF, solubilization of acetone precipitate etc.?

Thanks.
vic


it is difficult to precisely appreciate your problem; however, at first glance, I think a massive loss of your protein of interest is not to understand;

as the procedure for 2D is longer than for 1D, there should be thought of proteolytic degradation

-The Bearer-