proteins stuck at interface between stack and separating gel - (Jun/17/2009 )
My tech is having a problem that I cannot replicate or trouble shoot. We do Ni++ pulldowns in GuHCl, switch over to urea and then run on SDS-PAGE gradient gel. I've done this many times with many proteins and even used the same exact strains as he is using. In, seemingly random samples, his purified form gets stuck at the interface of the stack and separating gel. Not all of his lanes get stuck and sometimes not 100% of the purified protein, sometimes some of the protein migrates while some gets stuck. ALSO!! The background bands are there in the separating gel but the purified form gets stuck.
Does anyone have ANY idea what is going on??!!
change in conformation/size or charge of the protein of interest by incomplete removal of the Ni2+?
aggregated protein, random incomplete denaturing of aggregate (why some are okay and others aren't).