Re: electroelution of proteins
L. Castellanos-Serra (protchem@cigb.edu.cu)
Mon, 18 May 1998 09:28:57 EST
Dear Amanda
Working at the low picomol level we get high yields by
this passive elution procedure: Stain the gel by negative staining (Cu, Zn or for higher sensitivity,
ImH-Zn). Chelate the metal (this is crucial!) by 2 x 5 min. incubation in Tris, 0.2-0.4M Gly, pH 8.0
(about 1 ml per band). Wash a few seconds with 1 ml water. Crush the gel band through a metal sieve placed in a 1 ml
syringe, to get about 32 micrometer particles. Incubate with moderate vortexing, 2 x
10 min in Tris-Gly or ammonium bicarbonate (30-50 mM, pH 8.0, do not add detergent at
all) and 1 x 0.5 min in water. Collect each time by centrifugation ( a few seconds).
Each time the elution volume should be about twice the volume of the crushed gel. Note
that this procedure does not work with CB stained bands, only with
negative stain.
Proteins reduced in bME before electrophoresis tend to reaggregate on gel causing low elution
yields; thus, load in a sample buffer without bME or fully reduce and alkylate the sample in 8M urea before
electrophoresis. (Ref. J. Prot. Chem., 16, 415-419, 1997, Electrophoresis, 17, 1564-1572, 1996)
Lila Castellanos-Serra
<protchem@.cigb.edu.cu>
Prot. Chem. Dept.
Div. Phys. Chem. CIGB
FAX: 537-218070
Havana Cuba