Protein Protocols Protein Protocols

242 Smith
tougher (and smaller) by equilibration in aqueous ethanol, say, 40% (v/v) ethanol in water,
1 h or so. Too much ethanol may cause the gel to become opaque, but if this occurs merely
rehydrate the gel in a lower % (v/v) ethanol solution. A gradient gel may assume a slightly
trapezoid shape upon shrinkage in ethanol solutions—this makes scanning tracks down
the length of the gel more difficult. When scanning, eliminate dust, trapped air bubbles,
and liquid droplets, all of which contribute to noisy baseline.
17. Methods have been described for quantification of submicrogram amounts of proteins that
have been transferred from gels to polyvinylidene difluoride or similar membrane (e.g.,
7). A Sypro dye blot stain equivalent to the method described above for gels is available
from Molecular Probes. Note, however, that transfer from gel to membrane need not
(indeed, usually does not) proceed with 100% yield, so that results do not necessarily
accurately reflect the content of the original sample.
References
1. Dráber, P. (1991) Quantification of proteins in sample buffer for sodium dodecyl sulfatepolyacrylamide
gel electrophoresis using colloidal silver. Electrophoresis 12, 453–456.
2. Henkel, A. W. and Bieger, S. C. (1994) Quantification of proteins dissolved in an electrophoresis
sample buffer. Analyt. Biochem. 223, 329–331.
3. Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehhardt, W., and Taube, D. (1990) Essential
problems in quantification of proteins following colloidal staining with Coomassie
Brilliant Blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11, 101–117.
4. Syrovy, I. and Hodny, Z. (1991) Staining and quantification of proteins separated by polyacrylamide
gel electrophoresis. J. Chromatogr. 569, 175–196.
5. 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.
Analyt. Biochem. 213, 206–212.
6. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975) The use of Coomassie Brilliant Blue
G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on
polyacrylamide gels. Analyt. Biochem. 64, 509–516.
7. O’Keefe, D. O. (1994) Quantitative electrophoretic analysis of proteins labeled with
monobromobimane. Analyt. Biochem. 222, 86–94.
8. 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 immune-blotting. Analyt. Biochem. 220, 324–335.