PMID: 7535984Jan 1, 1995Paper

Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels

Analytical Biochemistry
C Fernandez-PatronL Castellanos-Serra

Abstract

A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring sin...Continue Reading

Citations

Jun 9, 2000·Molecular Microbiology·P R JungblutT F Meyer
May 8, 2008·The FEBS Journal·Tetsuro SamataTakahiro Akiyama
Apr 22, 2004·The Biochemical Journal·Mark E GrahamPeter L Jeffrey
Nov 22, 2002·The Journal of Biological Chemistry·Sebastian FrankenJoachim Kappler
Mar 18, 2006·Journal of Animal Science·R LametschP Ertbjerg
Jan 30, 2008·Current Protocols in Cell Biology·E C Dell'Angelica, J S Bonifacino
Apr 5, 2007·Nature Protocols·Lila Castellanos-Serra, Eugenio Hardy
Feb 26, 2014·The Biochemical Journal·Ricardo Soares-dos-ReisCarlos Reguenga
Jun 21, 2011·Journal of Chemical Biology·Victoria J GauciJens R Coorssen
Mar 3, 2010·Applied Biochemistry and Biotechnology·Mingri ZhaoXueqin Gong
Nov 19, 2015·Oral Diseases·P A van der WielenR D Cannon
Nov 5, 1999·International Immunology·R JordensF Koning
Sep 10, 1998·Biochemical and Biophysical Research Communications·C G ParkerH S Lichenstein
May 23, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Wayne F Patton
Jul 3, 1999·Clinical and Diagnostic Laboratory Immunology·E WedegeW D Zollinger
Sep 10, 2003·Biochemical and Biophysical Research Communications·Sreelatha T ReddyNancy M Dahms
Dec 1, 2004·Meat Science·R LametschE Bendixen
Aug 4, 2004·International Journal of Antimicrobial Agents·J Behra-MielletL Dubreuil

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