Protein Protocols Protein Protocols

Semidry Protein Blotting 333
24 h later the proteins with colloidal gold, we observed important contaminating spots on
the blots.
Only one point should be kept in mind when storing blots in PBS–azide. Sodium azide
inhibits peroxidase activity and therefore good washing of the membrane with PBS is
necessary before blot analysis using probe labeled with peroxidase.
As an alternative to blot storage, the gel can be frozen at –80°C. Immediately after electrophoresis,
the gel should be rinsed in distilled water for a few seconds and placed in a
plastic bag between two precooled glass plates. It is important to precool glass plates at
–80°C otherwise the gel will crack when thawed. These frozen gels can be stored at –80°C
for at least 3 mo (19). Before the transfer procedure, the frozen gels are thawed in their
plastic bags and then equilibrated as described in Subheading 3.
References
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(Baldo B. A. and Tovey, E. R., eds.), Karger, Basel, Switzerland, pp. 5–42.
2. Gershoni, J. M. (1985) Protein blotting: developments and perspectives. TIBS 10, 103–106.
3. Eckerskorn, C., Strupat, K., Schleuder, D., Hochstrasser, D. F., Sanchez, J. C., Lottspeich, F.,
and Hillenkamp, F. (1997) Analysis of proteins by direct scanning-infrared-MALDI mass
spectrometry after 2-D PAGE separation and electroblotting. Analyt. Chem. 69, 2888–2892.
4. Beisiegel, U. (1986) Protein blotting. Electrophoresis 7, 1–18.
5. Gershoni, J. M. and Palade, G. E. (1983) Protein blotting: principles and applications.
Analyt. Biochem. 131, 1–15.
6. Towbin, H. and Gordon, J. (1984) Immunoblotting and dot immunobinding: current status
and outlook. J. Immunol. Meth. 72, 313–340.
7 Wilkins, M. R. and Gooley, A. A. (1997) Protein identification in proteome projects, in
Proteomic Research: New Frontiers in Functional Genomics (Principles and Practice)
(Wilkins, M. R., Williams, K. L., Appel, R. D., Hochstrasser, D. F., eds.), Springer Verlag,
Berlin, pp. 35–64.
8. Gravel, P., Golaz, O., Walzer, C., Hochstrasser, D. F., Turler, H., and Balant, L. P. (1994)
Analysis of glycoproteins separated by two-dimensional gel electrophoresis using lectin
blotting revealed by chemiluminescence. Analyt. Biochem. 221, 66–71.
9. Sanchez, J. C., Ravier, F., Pasquali, C., Frutiger, S., Bjellqvist, B., Hochstrasser, D. F., and
Hughes, G. J. (1992) Improving the detection of proteins after transfer to polyvinylidene
difluoride membranes. Electrophoresis 13, 715–717.
10. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl.
Acad. Sci. USA 76, 4350–4354.
11. Erickson, P. F., Minier, L. N., and Lasher, R. S. (1982) Quantitative electrophoretic transfer
of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for
their re-use in immunoautoradiographic detection of antigens. J. Immunol. Meth. 51, 241–249.
12. Dunn, S. D. (1986) Effects of the modification of transfer buffer composition and the renaturation
of proteins in gels on the recognition of proteins on Western blots by monoclonal
antibodies. Analyt. Biochem. 157, 144–153.
13. Bjerrum, O. J. and Schafer-Nielsen, C. (1986). Buffer systems and transfer parameters for
semidry electroblotting with a horizontal apparatus, in Electrophoresis 1986 (Dunn, M. J.,
ed.) VCH, Weinheim, pp. 315–327.
14. Gershoni, J. M. and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium
dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Analyt.
Biochem. 124, 396–405.