SDS-PAGE hi pH/hi Tris loading buffer runs funny - Do proteins generally run funny on Lammelli SDS-PAGE with high pH/high (Feb/07/2007 )
I'm wondering if anyone has experience with running Lamelli gels using a loading buffer that has a high pH and high conc. of Tris.
A purified protein that is usually quite well-behaved showed abnormally high mobility by SDS-PAGE today. I resuspended a TCA ppt in 1x SDS-PAGE sample buffer (4% Ficoll, 2.5% SDS, 50mM Tris pH 6.8, 50mM DTT) and added Tris base to bring the color to blue. The final conc. of Tris in the sample was 200mM. In a standard Lammelli SDS-PAGE using buffers described below, the protein ran much faster than it should have. It normally runs at ~120kD, but on this gel it ran at 55kD.
Any ideas on whether the high conc. of Tris or the elevated pH of the sample buffer could have caused this fast migration?
Stacking gel: 125mM Tris pH 6.8, 0.1% SDS, 5% 37.5:1 bis:acrylamide
Resolving gel: 375mM Tris pH 8.8, 0.1% SDS, 10% 37.5:1 bis:acrylamide
Running buffer: 25mM Tris base, 0.192M glycine, 0.1% SDS
maybe you could check out the aggregative status of your protein. if it is a multimer, DTT may break down the disulfur-bond so that it becomes monomer. in that case, it would run much faster definitely.