Abstract
Negative staining of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels with salts of transition metals such as Cu2+ (Lee 1987), Ni2+ (Dzandu, 1988) and Zn2+ (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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Similar content being viewed by others
References
Castellanos-Serra, L., Fernández-Patrón, C., Hardy, E., Huerta, H. (1996) A procedure for protein elu-tion from reverse stained Polyacrylamide gels applicable at the low picomole level: an alternative route to the preparation of low abundance proteins for microanalysis. Electrophoresis 17:1564–1572
Castellanos-Serra, L., Fernández-Patrón, C., Hardy, E., Santana, H., Huerta, V. (1997) High yield elu-tion of proteins from sodium dodecylsulfate-polyacrylamide gels at the low—picomole level. Application to N-terminal sequencing of a scarce protein and to in solution biological activity analysis of on-gel renatured proteins. J. Prot. Chem. 16:415–419
Castellanos-Serra, L., Proenza, W., Huerta, V., Moritz, R. L., Simpson, R. J. (1999) Proteome Analysis of Polyacrylamide Gel Separated Proteins Visualized by Reversible Negative Staining Using Imidazole-Zinc Salts. Electrophoresis 20:732–737
Cohen, S. L., Chait, B. T. (1997) Mass spectrometry of whole proteins eluted from sodium docecyl-sulfate-polyacrylamide gels. Anal. Biochem. 247:257–267
Dzandu, J., Johnson, F., Wise, G. E. (1988) Sodium dodecylsulfate-gel electrophoresis: Staining of polypeptides using heavy metal salts. Anal. Biochem. 174:157–167
Fernández-Patron, C., Calero, M., Rodríguez, P. C, García, J. R., Musacchio, A., Soriano, F., Estrada, R., Frank, R., Castellanos-Serra, L., Méndez, E. (1995) Protein Reverse-staining: High-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224: 203–211
Fernández-Patrón, C., Castellanos-Serra, L. (1990) Presented at the Eight International Conference on Methods on Protein Sequence Analysis, July 1–6, (Abstract Booklet), Kiruna, Sweden.
Fernández-Patrón, C., Castellanos-Serra, L., Hardy, E., Guerra, M., Estévez, E., Mehl, E., Frank, R.W. (1998) Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: Generalization of the Reverse-staining technique. Electrophoresis 19:2398–2406
Fernández-Patrón, C., Castellanos-Serra, L., Rodríguez, P. (1992) Reverse-staining of sodium dode-cylsulfate Polyacrylamide gels by imidazole-zinc salts: Sensitive detection of unmodified proteins. BioTechniques 12: 564–573
Fernández-Patrón, C., Hardy, E., Sosa, A., Seoane, J., Castellanos-Serra, L. (1995) Double staining of Coomassie blue stained gels by imidazole-zinc Reverse-staining: sensitive detection of Coomassie blue undetected proteins. Anal. Biochem. 224:263–269
Gevaert, K., Demos, H., Sklyarova, T., Vandekerckhove, J., Houthaeve, T. (1998) A peptide concentration and purification method for protein characterization in the subpicomole range using MALDI-PSD sequencing. Electrophoresis 19: 909–1017
Hardy, E., Pupo, E., Casalvilla, R., Sosa, A., Trujillo, L., López, E., Castellanos-Serra, L. (1996) Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids by zinc-imidazole. Electrophoresis 17:1537–1541
Hardy, E., Pupo, E., Castellanos-Serra, L., Reyes, J., Fernández-Patrón, C. (1997) Sensitive Reverse-staining of bacterial lipopolysacharides on polyacrylamide gels by using zinc and imidazole salts. Anal. Biochem. 244:28–32
Hardy, E., Santana, H., Sosa, A., Hernández, L., Fernández-Patrón, C., Castellanos-Serra, L. (1996) Recovery of biologically active proteins detected with imidazole-SDS-zinc (Reverse-staining) on sodium dodecylsulfate gels. Anal. Biochem. 240:150–152
Heukeshoven, J., Dernick, R. (1991) Presented at the Electrophoresis Forum’ 91 (Munich), Forum Communication 501–506
Jensen, O.N., Podtelejnikov, A.V., Mann, M. (1997) Identification of the components as simple protein mixtures by high accuracy peptide mass mapping and database searching. Anal. Chem. 69:4741–4750
Lee, C., Levin, A., Branton, D. (1987) Cooper staining: A five-minute protein stain for sodium dodecylsulfate-polyacrylamide gels. Anal. Biochem. 166:308–312
Matsui, N.M., Smith, D.M., Clauser, K.R., Fichmann, J., Andrews, L.E., Sullivan, CM., Burlingame, A.L., Epstein, L.B. (1997), Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells. Electrophoresis, 18:409–417
Moritz, R.L. Simpson, R.J. (1992a) Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis. J. Chromatogr. 599:119–130
Moritz, R.L. & Simpson, R.J. (1992b) Purification of proteins and peptides for sequence analysis using microcolumn liquid chromatography. J.Microcol. Sep. 4:485–489
Moritz, R.L., Eddes, J.S., Reid, G.E., Simpson R.J. (1996b) S-pyridylethylation of intact Polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine-containing peptides. Electrophoresis. 17:907–917.
Moritz, R.L., Eddes, J.S., Reid, G.E., Simpson, R.J. (1996a) Capillary HPLC: A method for protein isolation and peptide mapping. Methods. 6:213–226.
Ortiz, M.L., Calero, M., Fernandez-Patron, C., Castellanos, L., Mendez, E. (1992) Imidazole-SDS-Zinc Reverse-staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins. FEBS Letters, 296:300–304
Reid, G.E., Rasmussen, R.K., Dorow, D.S., Simpson, R.J. (1998) Capillary column chromatography improves sample preparation for mass spectrometric analysis: complete characterization of human alpha- enolase from two-dimensional gels following in situ proteolytic digestion. Electrophoresis, 19:946–955
Scheler, C., Lamer, S., Pan, Z., Salnikov, J., Jungblut, P. (1998) Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix-assisted laser desorption/ionization-mass spectrometry. Electrophoresis 19:918–927
Zugaro, L.M., Reid G.E., Hong, Ji., Eddes J.S., Murphy, A.C., Burgess, A.W., Simpson R.J. (1998) Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry. Electrophoresis, 19:867–876.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Castellanos-Serra, L.R. et al. (2000). Reversible Negative Staining of Protein on Electrophoresis Gels by Imidazole-Zinc Salts: Micropreparative Applications to Proteome Analysis by Mass Spectrometry. In: Kamp, R.M., Kyriakidis, D., Choli-Papadopoulou, T. (eds) Proteome and Protein Analysis. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-59631-5_3
Download citation
DOI: https://doi.org/10.1007/978-3-642-59631-5_3
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-64060-5
Online ISBN: 978-3-642-59631-5
eBook Packages: Springer Book Archive