Protein Protocols Protein Protocols

428 Copse and Fowler
18. DDAO-phosphate should not be used with diethanolamine buffer, as it causes hydrolysis
of the substrate even in the absence of alkaline phosphatase.
19. The use of water between the scanning bed and the plastic wrap can reduce optical refraction
and give a clearer image.
4.3. Molecular Weight Markers
20. Protein molecular weight markers for use in Western blotting systems are usually
biotinylated, such that when incubated with streptavidin conjugated with AP or HRP, they
provide a ladder of bands, to give internal molecular weight standards. An alternative
system to this uses Perfect Protein TM Markers (Novagen), in which each protein contains
an S-Tag TM peptide that enables their detection on a blot using S-protein HRP or AP
conjugates in the secondary antibody or streptavidin incubation.
4.4. Time
21. The time required for detection of proteins by the chemifluorescence protocol described is
approx 5 h. Direct handling of the blots is usually minimal, meaning that it is possible to
run other experiments simultaneously if desired. However, if it is preferable to reduce
handling time further, the primary antibody itself may be HRP or AP conjugated, but this
may limit the sensitivity of detection considerably.
References
1. 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.
2. Renart, J., Reiser, J., and Stark, G. R. (1979) Transfer of proteins from gels to
diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody
specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76, 3116–3120.
3. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detection of DNAbinding
proteins by protein blotting. Nucleic Acids Res. 8, 1–20.
4. Gingrich, J. C., Davis, D. R., and Nguyen, Q. (2000) Multiplex detection and quantitation
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B. A., et al. (1998) Characterization of acridancarboxylic acid derivatives of chemiluminescent
peroxidase substrates. J. Org. Chem. 63, 930–937.
7. Appendix 2, Fluorescence Imaging: Principles and Methods (2000) Amersham Pharmacia
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9. Van Dam, A. (1994) Transfer and blocking conditions in immunoblotting, in Protein
Blotting–A Practical Approach (Dunbar, B. S., ed.), Oxford University Press, New York,
pp. 73–85.
10. Mansfield, M. A. (1994) Transfer and blocking conditions in immunoblotting, in Protein
Blotting–A Practical Approach (Dunbar, B. S., ed.), Oxford University Press, New York,
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milk. Analyt. Biochem. 181, 318–320.