Springer Protocols: Abstract: Staining of Preparative 2-D Gels: Coomassie Blue and Imidazole-Zinc Negative Staining

Staining of Preparative 2-D Gels: Coomassie Blue and Imidazole-Zinc Negative Staining

By: Neil M. Matsui², Diana M Smith-Beckerman³, Lois B. Epstein⁴

Abstract

Full Text

Download PDF (133K)

The identification of proteins using preparative gel electrophoresis and mass spectrometry requires reversible staining of relatively thick (1–1.5 mm) polyacrylamide gels. We have found that staining with colloidal Coomassie brilliant blue G-250 or negative staining with imidazole-zinc yields high-resolution stains (Fig. 1) that are compatible with subsequent mass spectrometric analysis (see Notes 1 and 5).

MediaObjects/978-1-59259-584-6_35_Fig1_HTML.jpg

Fig. 1. Preparative 2-D gels stained by the imidazole-zinc negative stain (left) and colloidal Coomassie blue G-250 stain (right). Gels were scanned by a Computing Densitometer (Molecular Dynamics, CA). Top: 1 mg of protein from ME-180 cervical carcinoma cells was separated by carrier ampholyte IEF and 11% SDS-PAGE, and then stained using the imidazole-zinc stain. Bottom: 1 mg of protein from A375 human melanoma cells was separated by carrier ampholyte IEF and 11% SDS-PAGE, and then stained using the colloidal Coomassie blue G-250.

Affiliation(s): (2) Department of Hematology, SFGH, San Francisco, CA
(3) Center for Biomedical Science, San Francisco State University, San Francisco, CA
(4) Cancer Research Institute and the Department of Pediatrics, University of California, San Francisco, CA

Book Title: 2-D Proteome Analysis Protocols

Series: Methods in Molecular Biology | Volume: 112 | Pub. Date: Sep-24-1998 | Page Range: 307-311 | DOI: 10.1385/1-59259-584-7:307

Subject: Biochemistry

References

Download References

1.	de Moreno, M. R., Smith, J. F., and Smith, R. V. (1986) Mechanism studies of coomassie blue and silver staining of proteins. J. Pharm. Sci. 75, 907–911.

2.	Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background and nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.

3.	Ortiz, M. L., Calero, M., Fernandez-Patron, C., Patron, C. F., Castellanos, L., and Mendez, E. (1992) Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins. FEBS Lett. 296, 300–304.

4.	Ferreras M., Gavilanes J. G., and Garcia-Segura, J. M. (1993) A permanent Zn²⁺ reverse staining method for the detection and quantification of proteins in polyacrylamide gels. Anal. Biochem. 213, 206–212.

5.	Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P. (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins. Biotechniques 12, 564–573.

6.	Smith, D. M., Tran, H. M., and Epstein, L. B. (1995) Two dimensional gel electrophoresis for purification, sequencing and identification of cytokine-induced proteins in normal and malignant cells, in Cytokines: A Practical Approach, 2nd ed. (Balkwill, F. R., ed.), IRL Press at Oxford University Press, Oxford, pp 111–128.

7.	Epstein, L. B., Smith, D. M., Matsui, N. M., Tran, H. M., Sullivan, C., Raineri, I., et al. (1996) Identification of cytokine-regulated proteins in normal and malignant cells by the combination of 2D-PAGE, mass spectrometry, Edman degradation, and immunoblotting: Approaches to analysis of their functional roles. Electrophoresis 17, 1655–1670.

8.	Matsui, N. M., Smith, D. M., Clauser, K. R., Fichmann, J., Andrews, L. E., Sullivan, C., et al. (1997) Immobilized pH gradient two dimensional gel electrophoresis and mass spectrometry identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells. Electrophoresis 18, 409–417.

9.	Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173–179.

10.	Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem. 68, 850–858.

Comments (Loading...)

Post comment/View all comments