Protein Protocols Protein Protocols

Production of Anitbodies Using Proteins 941
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Production of Antibodies Using Proteins in Gel Bands
Sally Ann Amero, Tharappel C. James, and Sarah C. R. Elgin
1. Introduction
A number of methods for preparing proteins as antigens have been described (1).
These include solubilization of protein samples in buffered solutions (ref. 2 and see
Chapter 128), solubilization of nitrocellulose filters to which proteins have been
adsorbed (ref. 3 and see Chapter 130), and emulsification of protein bands in polyacrylamide
gels for direct injections (4–8). The latter technique can be used to immunize
mice or rabbits for production of antisera or to immunize mice for production of monoclonal
antibodies (9–11). This approach is particularly advantageous when protein
purification by other means is not practical, as in the case of proteins insoluble without
detergent. A further advantage of this method is an enhancement of the immune
response, since polyacrylamide helps to retain the antigen in the animal and so acts as
an adjuvant (7). The use of the protein directly in the gel band (without elution) is also
helpful when only small amounts of protein are available. For instance, in this laboratory,
we routinely immunize mice with 5–10 µg total protein using this method; we
have not determined the lower limit of total protein that can be used to immunize rabbits.
Since polyacrylamide is also highly immunogenic, however, it is necessary in
some cases to affinity-purify the desired antibodies from the resulting antiserum or to
produce hybridomas that can be screened selectively for the production of specific
antibodies, to obtain the desired reagent.
2. Materials
1. Gel electrophoresis apparatus; acid-urea polyacrylamide gel or SDS-polyacrylamide gel.
2. Staining solution: 0.1% Coomassie brilliant blue-R (Sigma, St. Louis, MO, B-7920) in
50% (v/v) methanol/10% (v/v) acetic acid.
3. Destaining solution: 5%-(v/v) methanol/7% (v/v) acetic acid.
4. 2% (v/v) glutaraldehyde (Sigma G-6257).
5. Transilluminator.
6. Sharp razor blades.
7. Conical plastic centrifuge tubes and ethanol.
8. Lyophilizer and dry ice.
9. Plastic, disposable syringes (3- and 1-mL).
10. 18-gage needles.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
941