SDS-PAGE: Small protein diffuse and not staining well - (Nov/26/2007 )
I'm having an issue and I was hoping I might get some good advice here. I am currently expressing WT Calmodulin (CaM) from a pET15b plasmid in BL21(DE3) cells. I'm getting decent expression and that really isn't the issue. The problem I am having arises when I run sample from my cultures on gels to visualize expression. I have tried running 4-20% gradient gels, 15%, and 16% Tricine gels and in every case I get the same result. I see the induction of CaM in my induced cultures, however the CaM appears diffuse and the intensity of the band seems to decrease more so than the other bands upon destaining (I'm using a Coomassie G-250 staining protocol). I'm confident I am not overloading the lanes because when I load a control (purified CaM that I purchased) and the same thing happens - i.e. the band is diffuse and the protein doesn't hold the stain very well. This really isn't an issue in my experimental progress as I can visualize induction however it would be nice to get a clean sharp induction band for some figures. CaM is small protein (17kda).
Does anybody have any suggestions to fix this problem? I've thought about spiking the SLB with some EDTA but I haven't tried that.
I would appreciate any advice!
You may want to cross-link/fix the gel with 0.1 % glutaldehyde.
My lab routinely expresses proteins in the 10kD range without any problems resolving them clearly on a gel. We recently switched to 10-20% gels, but instead of Coomassie, we silver stain everything. If you're in love with coomassie (eventhough they aren't nearly as pretty as silver stains ), then try fixing the protein before Coomassie (we use formalin in our silver stains).
i think a normal 15%sds PAGE should work. I run an 11 K DA protein in this gel and of course stop the run before the dye front runs out.
we used 20% gels. let the gel run on 3/4 the way down. fix before staining with coomassie.
Let the gel polymerize for at least 3 hours.