I would suggest the zinc stain method, followed by any colormetric protein
concentration determination method. BioRad Zinc Stain is a good
commercial one. I have also had good luck with the BioRad Bradford-based
dye binding assay, or the DC version of it for proteins in detergent. The
BCA
should work too. though.
I am assuming you don't have extinction coefficients
for these proteins to measure their concentration directly by UV? That would
be my first choice, if I knew (or could determine) the Em.
I also assume you are running the gels long enough to adequately resolve
these
adjacent bands. We routinely use prestained markers in one lane of the gel
to
monitor the electrophoresis real-time, which allows you to run the dye front
off of
the gel to improve the resolution at higher molecular weights. That way we
can
get maximum recovery of both proteins with minimum cross-contamination.
Nadine Ritter
Abbott Diagnostics Division
Nadine.Ritter@add.ssw.abbott.com
____________________________________________
I am purifying two proteins that are quite close on gels. I stain the gel
with coomassie, cut out the proteins and electroelute. I would like to
measure protein content on the eluate but most assays are based on
coomassie so they won't work. Can anyone tell me if the BCA assay would
do it? We have used an assay that depends on the size of the protein spot
on a membrane and find it is not very reliable.
I have tried staining the gel with copper but the bands are difficult to
distinguish. Would zinc stain be better?
Thank you.
Helene Z Hill, Ph.D. Internet: hill@umdnj.edu
Head, Section of Cancer Biology Voice/fax: (973)972-3421
Mail: MSB-E586
NJ Medical School
185 South Orange Ave
Newark, NJ 07103-2714