Re: electroelution of proteins
Gautam Sarath (gsarath@unlinfo.unl.edu)
Sun, 17 May 1998 10:35:30 -0500 (CDT)
Dear Amanda: Over the last decade or so, my success with electroelution of
proteins from SDS-PAGE have never been too good. Although, it is generally
better after Native PAGE or IEF (the losses are still high). In my hands,
passive elution of unstained bands after SDS-PAGE is almost quantitative
and works better than any electroelution protocol. One would expect that
this type of system (electroelution) should work, and I have not figured
out what the problem could be, however, if one were to set up a system for
the downward electrotransfer of proteins (aka-blots) it should give much
higher yields. I wonder if this transfer is related to the actual
cross-sectional volume through which transfer proceeds: eg, if an agarose
gel is submerged in a very large voulme of buffer, there is essentially a
very small current through the gel). Ideally electrotransfer should be
enhanced if the electrodes are close together and the total distance for
protein movement is small. Although one can elute stained-bands,
recoveries are even more compromized. I have also had poor success using
negative-staining methods. Best results were seen when the gel was run
with a fluorescent marker appropriate to the band of interest, then slicing
the gel lanes at the required region, mashing the gel pieces into water or
a buffer (about 2 to 4 X volume) and passive-eluting at 4C overnight on a
tumbler-shaker . Spin the gelpieces out (filter) and the liquid can be
lyophilized or concentrated by the method of Wessel & Fluuge, 1984,
Analytical Biochem). This works for protein loads from about 0.1ug
onwards, but we never done a quantitative test, maybe I can get some real
numbers and post it on this site. Hope this helps in some form, Gautam
Gautam Sarath
N-226, Beadle center
Protein Core Facility
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842