Zinc/Imidazole Procedure for Visualization of Proteins in Gels ...
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Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4701
Protocol
Zinc/Imidazole Procedure for Visualization of Proteins in Gels by Negative Staining
Richard J. Simpson
This protocol was adapted from "Peptide Mapping and Sequence Analysis of Gel-Resolved Proteins," Chapter 7, in Proteins and Proteomics by Richard J. Simpson. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, USA, 2003.
INTRODUCTION
The zinc/imidazole staining procedure for visualizing proteins in acrylamide gels is based on differential salt binding. Because protein-bound salts (e.g., dodecyl sulfate or the heavy
cation zinc) are chemically less active than free zinc ions in the gel, precipitation of an insoluble salt is much slower in those regions of the gel occupied by proteins than in the gel
background where zinc dodecyl sulfate precipitates. The result is a "negative stain," with translucent proteins and an opaque gel background, due to zinc dodecyl sulfate precipitation.
The sensitivity of the method has been markedly improved by altering the composition of the precipitated salt to a complex of zinc and imidazole. This protocol provides two methods:
reverse stain using imidazole, SDS, and zinc, and double-staining using Coomassie blue stain followed by zinc/imidazole.
RELATED INFORMATION
The protocol described here was adapted from Fernandez-Patron et al. (1995a,b) and Simpson and Reid (1998).
MATERIALS
Reagents
Fixing solution for negative staining (for Procedure A)
Imidazole-SDS solution
Protein samples within a gel that have been separated by electrophoresis
These proteins should be either unstained (Procedure A) or stained with Coomassie Brilliant Blue (Procedure B; see Staining Proteins in Gels with Coomassie Blue for staining
procedure).
Zinc sulfate (0.2 M)
Equipment
Containers for incubating the gel
Shaker
METHOD
Use either Procedure A (Steps 1-5) or Procedure B (Steps 6-9).
Procedure A: Direct Reverse Staining with Imidazole, SDS, and Zinc
1. Submerge the gel in fixing solution for 20 minutes with gentle shaking.
2. Discard the fixing solution. Wash the gel twice in H2O for 15 minutes with gentle shaking.
3. Incubate the gel in the imidazole-SDS solution for 15 minutes with gentle shaking.
4. Remove the imidazole-SDS solution. Add 0.2 M zinc sulfate to the gel and agitate it for 30-60 seconds.
5. When the gel has stained satisfactorily, remove the zinc sulfate solution and submerge the gel in H2O.
Procedure B: Double Staining of Coomassie-Blue-Stained Polyacrylamide Gels with Imidazole, SDS, and Zinc
6. After Coomassie blue staining (see Staining Proteins in Gels with Coomassie Blue), wash the destained gel twice in H2O for 15 minutes with gentle shaking.
7. Incubate the gel in the imidazole-SDS solution for 15 minutes with gentle shaking.
8. Remove the imidazole-SDS solution. Add 0.2 M zinc sulfate to the gel, and agitate it for 30-60 seconds.
9. When the gel has stained satisfactorily, remove the zinc sulfate solution and submerge the gel in H2O.
DISCUSSION
Typical protein detection sensitivities with this zinc/imidazole staining procedure approach the low-nanogram range. The stain is reversible when divalent chelators, such as EDTA, are
included. The method can also be adapted to proteins blotted onto membranes (Patton et al. 1994) and to general proteome analysis of gel-resolved proteins (Castellanos-Serra et al.
1999).
REFERENCES
1. Castellanos-Serra, L., Proenza, W., Huerta, V., Moritz, R.L., and Simpson, R.J. 1999. Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible
negative staining using imidazole-zinc salts. Electrophoresis 20: 732–737.[Medline]
2. Fernandez-Patron, C., Calero, M., Collazo, P.R., Garcia, J.R., Madrazo, J., Musacchio, A., Soriano, F., Estrada, R., Frank, R., Castellanos-Serra, L.R., et al. 1995a. Protein
reverse staining: High-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal Biochem 224: 203–211.[Medline]
3. Fernandez-Patron, C., Hardy, E., Sosa, A., Seoane, J., and Castellanon, L. 1995b. Double staining of Coomassie blue-stained polyacrylamide gels by imidazole-sodium dodecyl
sulfate-zinc reverse staining: Sensitive detection of Coomassie blue-undetected proteins. Anal Biochem 224: 263–269.[Medline]
4. Patton, W.F., Lam, L., Su, Q., Lui, M., Erdjument-Bromage, H., and Tempst, P. 1994. Metal chelates as reversible stains for detection of electroblotted proteins: Application to
protein microsequencing and immunoblotting. Anal Biochem 220: 324–335.[Medline]
5. Simpson, R. and Reid, G.E. 1998. Sequence analysis of gel-resolved proteins. In Gel electrophoresis of proteins: A practical approach, 3rd ed. (ed. B.D. Hames), pp. 237–267.
Oxford University Press, Oxford, UK.
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Zinc/Imidazole Procedure for Visualization of Proteins in Gels ...
http://cshprotocols.cshlp.org.libproxy.mit.edu/cgi/content/full/2...
Caution
General warning
This material contains hazardous components. Please see recipe for full details.
Caution
Zinc sulfate (ZnSO4)
Zinc sulfate (ZnSO4) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.
Recipe
Fixing solution for negative staining
Methanol (50% [v/v])
Glacial acetic acid (5% [v/v])
H2O (45%)
Recipe
Imidazole-SDS solution
6.8 g imidazole
0.5 g SDS
Dissolve the imidazole and SDS in 500 ml H2O. The final concentrations are 0.2 M imidazole and 0.1% SDS.
Recipe
Zinc sulfate (0.2 M)
28.7 g ZnSO4·7H2O
Dissolve the zinc sulfate in 500 ml H2O.
Copyright © 2007 by Cold Spring Harbor Laboratory Press. Terms of Service. All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties
with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with
caution. For a full listing of cautions, click here.
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