Protein Protocols Protein Protocols

952 Diano and Le Bivic
tual excess of antigen solution is then injected into the back of the rabbit, as described in
Methods. The boosters are given in the classic manner, that is, in several subcutaneous
injections into the rabbit’s back. If the amount of protein available is even more limited, a
guinea pig may be immunized (first immunization in the Iymphatic ganglion, and boosters,
as usual).
15. The advantage of getting a high titer for the antibody of interest is that the amount of
irrelevant antibodies is, by comparison, very low and, consequently, does not generate
any background. Another advantage of using a crude serum with a high antibody titer is
that this serum may be used without further purification to screen a cDNA expression
library (12).
16. The time required for transfer and the intensity used are dependent on the molecular weight
and the nature of the protein to be transferred (hydrophilic or hydrophobic). During transfer,
the electrophoresis tank may be cooled with tap water. We, however, prefer semidry
transfer, which is time and buffer saving and cooling is not necessary.
References
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3. O’Farrel, P. Z., Goodman, H. M., and O’Farrel, P. H. (1977) High resolution two-dimensional
electrophoresis of basic as well as acidic proteins. Cell 12, 1133–1142.
4. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (Lond.) 227, 680–685.
5. Görg, A. (1998) A laboratory manual. http://www.weihenstephan.de/blm/deg.
6. Knudsen, K. A. (1985) Proteins transferred to nitrocellulose for use as immunogens. Analyt.
Biochem. 147, 285–288.
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polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody
and radioiodinated protein A. Analyt. Biochem. 112, 195–203.
8. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1971) A method for producing
specific antisera with small doses of immunogen. J. Clin. Endocrinol. 33, 98–988.
9. Dunn, M. J. and Patel, K. (1988) 2-D PAGE using flat bed IEF in the first dimension, in
Methods in Molecular Biology, Vol. 3: New Protein Techniques (Walker, J. M., ed.),
Humana Press, Totowa, NJ, pp. 217–232.
10. Dunn, S. D. (1986) Effects of the modification of transter 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.
11. Sigel, M. B., Sinha, Y. N., and Vanderlaan, W. P. (1983) Production of antibodies by
inoculation into lymph nodes. Meth. Enzymol. 93, 3–12.
12. Preziosi, L., Michel, G. P. F., and Baratti, J. (1990) Characterisation of sucrose hydrolizing
enzymes of Zymomonas mobilis. Arch. Microbiol. 153, 181–186.