We had the same problem. We attributed it to proteolysis. It is easy to
contaminate things, because bacteria and fungi grow in everything. Make
buffers fresh with deionized, sterilized water, clean glass ware just
before use, rinsing with deionized, sterilized water. Add fresh protease
inhibitors to your extracts, just before swelling the strips. As a last
resort, boil your extracts for five minutes or so before loading. If you
want to use stock solutions, aliquot in individual amounts and freeze.
Wear gloves for everything and don't walk around touching other things with
the gloves. don't recycle the mineral oil. Get the cleanest agarose to
seal the IEF to the SDS-PAGE. Pick a relatively dust-free area to work in.
Katheryn REsing
On Mon, 26 Jan 1998 at 11:49:03 Judith A. Airey wrote:
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