I am having a problem with efficiency of IEF. When I stain the second
dimension there is much less protein visible than the imput amount -
somewhere in the region of 20% recovery. I make a well in the second
dimension and load some of the imput sample as a standard. The system I
am using is the Pharmacia system with dry strips for the focussing. The
strips are both commercial and ones I have made myself. The second
dimension is standard SDS PAGE. I have tried membrane preps, molecular
weight standards and individual proteins such as BSA. As suggested by
several people I have tried different detergents and different
concentrations, including CHAPS and Triton (with an initial solubilization
with SDS for membrane proteins). I have used urea with and without the
addition of thiourea. I have tried varying the equilibration solution
between the first and second dimension, including increasing the SDS
concentration. This problem seems to be independent of pI.
So, I am wondering where is the rest of my protein? Am I only focussing
20% of the protein? It was suggested to me that the protein focusses but
not all the protein migrates into the second dimension. If that is the
case how do I get more protein out of the strip? I don't think I am
loosing that much protein out of the stip when I equilibrate between the
dimensions since there seems to be no relationship with size or
solubility, and recovery.
Has anybody else had this problem? I need to increase my efficiency since
I am limited in my initial sample size.
Thanks, Judith
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Judith A. Airey
Dept. Pharmacology/318
University of Nevada, Reno
Reno, NV 89557
Tel: 702 784 4651
Fax: 702 784 1620