CNBr and Tris/Tricine gels
Gautam Sarath (gsarath@unlinfo.unl.edu)
Fri, 14 Aug 1998 09:57:36 -0500 (CDT)
Dear Deb: Just last week I carried out a trial for three recombinant
proteins digested with CNBr. In our hands, we got really great results
with dried protein resuspended in 70 % formic acid and digested with CNBr,
1 to 3 mg/reaction. Samples digested from SDS-gel slices were not as clean.
The samples were speed-vapped and redired twice with water, resuspended in
1X Tris/Tricine loading buffer and electrophoresed on a gradient gel. I
purchased the premade gradient gels and buffers from BioRAD. They worked
very well and are worth the time saved in making Tris/Tricine gradient
gels. If the gels are fixed first in 50% methanol/10% acetic acid, most of
the small peptides are stained (down to about 3 kDa), I think a post
fixation in 10% glutaraldehyde will do the trick for even smaller peptide,
alternatively, a good transfer to PVDF will achieve the same results. I
would strongly reccomend the BioRAD stuff since it takes out the worry of
gel to gel variations, gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html