Reversible Negative Staining of Protein on Electrophoresis Gels
51
For the high-sensitivity separation of peptides for subsequent analysis, peptides obtained from in-gel digests of Znll Reversed-stained proteins can also be
separated by capillary RP-HPLC and collected manually into 1.5 ml polypropylene tubes (Moritz & Simpson, 1992b). Here, with the aid of DAD and the use of
second-derivative spectra, the identification of peptides containing aromatic residues or moieties can be readily identified. Fig. 3.6 shows a typical highsensitivity UV trace using DAD set at 215 nm of a capillary RP-HPLC separation
of a PA gel separated protein, using recombinant interleukin-6 (IL-6) as an example. IL-6 (SOOng) was electrophoresed on 4-20 % precast PA gel (Novex), and following visualization by imidazole-zinc Reverse-staining, subjected to in-gel
alkylation followed by digestion with trypsin. The resultant peptides (25 %, ~
5picomol) were fractionated by capillary RP-HPLC that was directly coupled to
the Hewlett-Packard fused-quartz capillary flow cell. The column effluent was
monitored at 5 different wavelengths (200nm, 215nm, 254nm, 280 nm and
290nm, data not shown) simultaneously with UV spectra between 240-320 nm
collected "on-the-fly" throughout the separation. The identification of a cysteinecontaining peptide was determined by monitoring the UV spectra and peptide
T1 was identified with a characteristic absorbance at 254 nm (Fig 3.6, inset).
3.3
Proteomics
In summary, the combined use of the high-sensitivity Znll Reverse-stain with
state-of-the-art mass spectrometry provides a powerful suite of methods for the
rapid purification, identification and characterization of low-abundance proteins
available. Additionally, these methods provide analysis without the complication
of additional contaminants and methods-based protein modification. With the
advent of automation, these procedures should be a powerful adjunct to proteorne analysis.
References
Castellanos-Serra, L., FernandeZ-Patron, c., Hardy, E., Huerta, H. (1996) A procedure for protein elution from reverse stained polyacrylamide gels applicable at the low picomole level: an alternative
route to the preparation oflow abundance proteins for microanalysis. Electrophoresis 17:1564-1572
Castellanos-Serra, L., Fernandez-Patron, c., Hardy, E., Santana, H., Huerta, V. (1997) High yield elution 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. Chern. 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 docecylsulfate-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
Fernandez-Patron, C., Calero, M., Rodriguez, P. c., Garcia, J. R., Musacchio, A., Soriano, F., Estrada,
R., Frank, R., Castellanos-Serra, L., Mendez, E. (1995) Protein Reverse-staining: High-efficiency
microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224:
203-211
Fernandez-Patron, c., Castellanos-Serra, L. (1990) Presented at the Eight International Conference on
Methods on Protein Sequence Analysis, July 1-6, (Abstract Booklet), Kiruna, Sweden.
51
For the high-sensitivity separation of peptides for subsequent analysis, peptides obtained from in-gel digests of Znll Reversed-stained proteins can also be
separated by capillary RP-HPLC and collected manually into 1.5 ml polypropylene tubes (Moritz & Simpson, 1992b). Here, with the aid of DAD and the use of
second-derivative spectra, the identification of peptides containing aromatic residues or moieties can be readily identified. Fig. 3.6 shows a typical highsensitivity UV trace using DAD set at 215 nm of a capillary RP-HPLC separation
of a PA gel separated protein, using recombinant interleukin-6 (IL-6) as an example. IL-6 (SOOng) was electrophoresed on 4-20 % precast PA gel (Novex), and following visualization by imidazole-zinc Reverse-staining, subjected to in-gel
alkylation followed by digestion with trypsin. The resultant peptides (25 %, ~
5picomol) were fractionated by capillary RP-HPLC that was directly coupled to
the Hewlett-Packard fused-quartz capillary flow cell. The column effluent was
monitored at 5 different wavelengths (200nm, 215nm, 254nm, 280 nm and
290nm, data not shown) simultaneously with UV spectra between 240-320 nm
collected "on-the-fly" throughout the separation. The identification of a cysteinecontaining peptide was determined by monitoring the UV spectra and peptide
T1 was identified with a characteristic absorbance at 254 nm (Fig 3.6, inset).
3.3
Proteomics
In summary, the combined use of the high-sensitivity Znll Reverse-stain with
state-of-the-art mass spectrometry provides a powerful suite of methods for the
rapid purification, identification and characterization of low-abundance proteins
available. Additionally, these methods provide analysis without the complication
of additional contaminants and methods-based protein modification. With the
advent of automation, these procedures should be a powerful adjunct to proteorne analysis.
References
Castellanos-Serra, L., FernandeZ-Patron, c., Hardy, E., Huerta, H. (1996) A procedure for protein elution from reverse stained polyacrylamide gels applicable at the low picomole level: an alternative
route to the preparation oflow abundance proteins for microanalysis. Electrophoresis 17:1564-1572
Castellanos-Serra, L., Fernandez-Patron, c., Hardy, E., Santana, H., Huerta, V. (1997) High yield elution 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. Chern. 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 docecylsulfate-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
Fernandez-Patron, C., Calero, M., Rodriguez, P. c., Garcia, J. R., Musacchio, A., Soriano, F., Estrada,
R., Frank, R., Castellanos-Serra, L., Mendez, E. (1995) Protein Reverse-staining: High-efficiency
microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224:
203-211
Fernandez-Patron, c., Castellanos-Serra, L. (1990) Presented at the Eight International Conference on
Methods on Protein Sequence Analysis, July 1-6, (Abstract Booklet), Kiruna, Sweden.