CHAPTER 3
Reversible Negative Staining of Protein
on Electrophoresis Gels by Imidazole-Zinc Salts:
Micropreparative Applications to Proteome Analysis
by Mass Spectrometry
L. R. CASTELLANOS-SERRA 1, E. HARDY!, W. PROENZA \ V. HUERTA!,
L. J. GONZALEZ!, J. P. LE-CAER Z , R. L. MORITZ 3 and R. J. SIMPSON 3
1
Introduction
Negative staining of proteins on sodium dodecyl sulfate {SDS)-polyacrylamide gels
with salts of transition metals such as Cu z + (Lee 1987), Ne+ (Dzandu, 1988) and Znz+
(Dzandu, 1988) was first introduced about 10 years ago. This ingenious type of staining is based on the selective precipitation of metal salts on the gel surface except
where proteins are located. Consequently, proteins appear in the gel as transparent,
colorless bands (or spots) against a slightly stained background. Unfortunately,
reproducibility and sensitivity are highly influenced by the electrophoresis - associated reagents remaining in the gel after completion of the electrophoretic run. Such
variability in the gel composition, that leads to differences in sensitivity and reproducibility, is particularly significant when scarce amounts of proteins are present.
In 1990 the group working at C.I.G.B in Havana improved the reproducibility
and sensitivity of the metal negative staining procedure by exchanging soluble
gel components (buffers, salts etc.,) with imidazole (ImH) prior to metal staining
(Fernandez-Patron and Castellanos-Serra 1990). Imidazole is well known in protein chemistry for its strong interaction with transition metals, a property that
has been largely exploited for the design of poly-histidine tail fused proteins for
immobilized metal affinity chromatography. Imidazole-zinc staining combines
several features that are highly appreciated for a detection procedure:
i) speed (15 mins or less) and simplicity (two steps),
ii) highly reproducible sensitivity in the femtomole range, closely approaching
that of conventional silver staining,
iii) it does not cause modifications of proteins,
iv) proteins are reversibly fixed in the gel through zinc-mediated complexes and
v) the staining reagents are neither toxic nor environmentally dangerous. This
detection procedure is based on the generation of an insoluble salt, zinc imidazolate (ZnIm2), in the gel matrix. The mechanism of negative staining has
been investigated, and the influence of common electrophoresis reagents
including SDS on gel staining, was determined. (Fernandez-Patron et al. 1998).
1 Center for Genetic Engineering and Biotechnology (CIGB) P.O. BOX 6162, Havana 10600, Cuba.
2 Ecole Superieure de Chimie et Physique de la Ville de Paris, 10 rue Vauquelin, Paris 75024 France.
3 Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne) and the
Walter and Eliza Hall of Medical Research, Parkville, Victoria, Australia.
Reversible Negative Staining of Protein
on Electrophoresis Gels by Imidazole-Zinc Salts:
Micropreparative Applications to Proteome Analysis
by Mass Spectrometry
L. R. CASTELLANOS-SERRA 1, E. HARDY!, W. PROENZA \ V. HUERTA!,
L. J. GONZALEZ!, J. P. LE-CAER Z , R. L. MORITZ 3 and R. J. SIMPSON 3
1
Introduction
Negative staining of proteins on sodium dodecyl sulfate {SDS)-polyacrylamide gels
with salts of transition metals such as Cu z + (Lee 1987), Ne+ (Dzandu, 1988) and Znz+
(Dzandu, 1988) was first introduced about 10 years ago. This ingenious type of staining is based on the selective precipitation of metal salts on the gel surface except
where proteins are located. Consequently, proteins appear in the gel as transparent,
colorless bands (or spots) against a slightly stained background. Unfortunately,
reproducibility and sensitivity are highly influenced by the electrophoresis - associated reagents remaining in the gel after completion of the electrophoretic run. Such
variability in the gel composition, that leads to differences in sensitivity and reproducibility, is particularly significant when scarce amounts of proteins are present.
In 1990 the group working at C.I.G.B in Havana improved the reproducibility
and sensitivity of the metal negative staining procedure by exchanging soluble
gel components (buffers, salts etc.,) with imidazole (ImH) prior to metal staining
(Fernandez-Patron and Castellanos-Serra 1990). Imidazole is well known in protein chemistry for its strong interaction with transition metals, a property that
has been largely exploited for the design of poly-histidine tail fused proteins for
immobilized metal affinity chromatography. Imidazole-zinc staining combines
several features that are highly appreciated for a detection procedure:
i) speed (15 mins or less) and simplicity (two steps),
ii) highly reproducible sensitivity in the femtomole range, closely approaching
that of conventional silver staining,
iii) it does not cause modifications of proteins,
iv) proteins are reversibly fixed in the gel through zinc-mediated complexes and
v) the staining reagents are neither toxic nor environmentally dangerous. This
detection procedure is based on the generation of an insoluble salt, zinc imidazolate (ZnIm2), in the gel matrix. The mechanism of negative staining has
been investigated, and the influence of common electrophoresis reagents
including SDS on gel staining, was determined. (Fernandez-Patron et al. 1998).
1 Center for Genetic Engineering and Biotechnology (CIGB) P.O. BOX 6162, Havana 10600, Cuba.
2 Ecole Superieure de Chimie et Physique de la Ville de Paris, 10 rue Vauquelin, Paris 75024 France.
3 Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne) and the
Walter and Eliza Hall of Medical Research, Parkville, Victoria, Australia.