Acid Native PAGE

lou sheridal at mrc-lmb.cam.ac.uk
Tue Nov 7 12:08:50 EST 2000


I have two proteins, one with a pI of 8.8 (Mw=50kDa) and the other with
a pI of 8.3 (Mw=90kDa) I am trying to make a complex of the two proteins
(which should be irreversible) and would like to see this complex
formation, preferably on a native gel.

I have attempted to run an acid native PAGE,. I have made my gels (25mM
Tris Glycine, 12% Acrylamide) and the buffer is Tris (25mM)  Alanine,
pH7.4. The buffer that the samples are in vary but all are 25mM buffer
with 100mM NaCl. The loading dye contains glycerol and methyl green.

The problem that I am having is that the samples do not run very far and
the bands are quite broad. I have heard that lowering the salt
concentration of the samples can help with resolution, but by how much.
Also does anyone know if the pH that I have tried is low enough?? Should
I just try another gel at lower pH?? Is there any other way in which
people might be able to advise me??

I hope that someone can help
Thanks

Lou



---






More information about the Proteins mailing list