I suspect that a stock reagent/buffer has been made incorrectly in the lab. I have tried to make everything fresh and the problem persisted. Anyone know what the culprit could be? I haven't been able to find anybody else in the lab, using the same reagents, that is having this problem.
My protein is 17 and 22kDa (endogenous and overexpressed versions). I am loading 50ug of protein, but this is the same as I have always used. I use 5x SDS sample buffer (containing 10% beta-mercapto) (currently using new aliquot of same frozen buffer, with beta mercapto added after thawing), I boil sample for 5-10mins at 95 degrees. I run on a 15% Tris-HCL SDS Gel. I have tried running at 60V, 80V, 100V, 110V, all resulting in the same problem. The standard also seems quite smeared in the low molecular weight range so I think that it is a gel problem rather than a transfer problem. But anyway, I transfer in 20% Methanol, Tris Glycine at 180Amp for 1.5Hr, onto prewet (in methanol) PVDF.
I have made fresh:
-the 10% APS ,
-the Tris-HCL electroporesis buffer (including 10% SDS, though this took a very long time to go into solution (~1hr)). Incidently, our pH meter is currently not working so I had to use one at a neighbouring lab, which perhaps is not calibrated correctly?
Perhaps it is a problem with the 10% SDS? pH? sample buffer? 2 week old bottle of acrylamide? 1 year old bottle of TEMED?
Somebody has suggested to me that it looks like protein degradation, but why would this have started occuring only now, and with samples that used to run fine and still run fine on a precast gel?
AAAAAAAAAAAAAAAAAAARGH, Any help will be greatly appreciated
Edited by Analyse, 02 October 2010 - 01:45 PM.