Seperating High MW proteins - (Jan/13/2009 )
I'm trying to get good separation of high MW proteins ranging from 150kDa to 500kDa.
I've been using 8% Tris-HCl SDS-PAGE with a 3% stacking gel, which I have to run for fairly long periods to allow the larger proteins to run into the gel. We use the Bio-rad proteanII minigel system, which has worked well for us so far.
I see that Tris-Acetate gels are recommended for separation for high MW proteins. I'd like to give this a go, but can't find a good protocol anywhere.
I know Invitrogen and Bio-rad sell pre-cast gels, but that's not an option, we have to pour our gels ourselves.
If anyone has anything links or protocols or thoughts, they'd be much appreciated.
this reference may contain the formulation you need:
J.V. Maizel, Jr., Polyacrylamide Gel Electrophoresis of Viral Proteins. In: K. Maramorosch and H. Koprowski, Editors, Methods in Virology Vol. 5, Academic Press, New York (1971)
(i have a copy that i can't locate right now so i can't check and post the formulation).
you can also try a gradient gel to sharpen and enhance the separation (3-8 should be okay). you can also try a 5% gel.
we used to separate myosin heavy chains with a 3.5-5% gradient with a 8-0M urea gradient (in sds).
bio-rad's tris-acetate gels are non-denaturing, are you sure that you want to try it?
Just overrun a 6% gel for 20 minutes. That should do yr job.
Chk this out: