A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis

Electrophoresis
L Castellanos-SerraV Huerta

Abstract

We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein stainin...Continue Reading

References

Mar 1, 1989·Analytical Biochemistry·R J SimpsonE C Nice
Jan 1, 1995·Analytical Biochemistry·C Fernandez-PatronL R Castellanos-Serra
Jan 1, 1995·Analytical Biochemistry·P JenöM Horst
May 15, 1994·Analytical Biochemistry·P ChenL W Dittert
Oct 1, 1996·Electrophoresis·E HardyL Castellanos-Serra

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Citations

Apr 19, 2005·Biotechnology Letters·Manuel MansurEduardo Martínez
Jun 21, 2011·Journal of Chemical Biology·Victoria J GauciJens R Coorssen
Jun 4, 1999·Progress in Biophysics and Molecular Biology·K A Resing, N G Ahn
May 23, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Wayne F Patton
Apr 5, 2007·Nature Protocols·Lila Castellanos-Serra, Eugenio Hardy
Oct 26, 2010·American Journal of Respiratory and Critical Care Medicine·Fernando SpillerFernando Q Cunha
Dec 1, 2007·Proceedings of the National Academy of Sciences of the United States of America·F L A C MestrinerF Q Cunha
Jul 18, 2012·Analytical Biochemistry·Guo-Qing LiCheng-Xi Cao
Sep 25, 2008·Journal of Proteomics·Louise N PatonWarren G Bryson
Sep 23, 2006·Proteomics·Ingrid MillerElisabetta Gianazza
Sep 11, 2004·Biochemical and Biophysical Research Communications·Syujirou MatsuyamaNobuhiro Sato
Apr 15, 2004·Analytical Biochemistry·Eugenio Hardy, Lila R Castellanos-Serra
May 30, 1998·Analytical Biochemistry·E HardyL R Castellanos-Serra
Mar 26, 1998·Analytical Biochemistry·L R Castellanos-SerraJ C Sánchez

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