Protein Protocols Protein Protocols

450 Kaufmann
erasure buffer (16). Incubation times for removal of antibodies after chemiluminescent
detection have not been systematically investigated.
18. Although most epitopes are resistant to the erasure procedure (Figs. 2 and 4B; see also
refs. 15,16), epitopes recognized by an occasional monoclonal antibody are destroyed by
erasure (Fig. 4A, lane 2). Observations from our laboratory also indicate that certain
epitopes are lost upon prolonged storage of blots (Fig. 4B, lane 3). There does not appear
to be any relationship between the loss of epitopes upon blot storage and the damage of
epitopes during the erasure procedure.
4.4. Removal of Antibodies After Autoradiographic Detection
19. 125I-labeled protein A can be substituted for radiolabeled secondary antibody. Protein A,
however, can bind to the immunoglobulins present in milk, causing a high background on
the blot. Therefore, when 125I-labeled protein A is to be used, milk should not be utilized
to block unoccupied binding sites (Subheading 3.1., step 11), nor as a diluent for antibodies
(Subheading 3.2., steps 2, 3, and 6). Instead, bovine serum albumin, hemoglobin, or
gelatin should be considered (see Note 6).
20. The major modification suggested with radiolabled secondary antibodies is a reincubation
of blots with protein-containing buffer prior to drying. For reasons that are unclear, this
step appears to be essential for efficient dissociation of certain antibodies from nitrocellulose
or PVDF after incubation with radiolabeled secondary antibodies (Fig. 3), but not
after incubation with peroxidase-coupled antibodies (Fig. 2).
4.5. General Notes
21. Some blotting procedures involve deposition of chromogenic reaction products directly
on immunoblots by enzyme-coupled secondary antibodies (5,23,24). The technique
described above is not useful for removing colored peroxidase reaction products (e.g.,
diaminobenzidine oxidation products) from blots.
22. A modification of the techniques described in Subheading 3.2. allows the detection of
glycoproteins by radiolabeled lectins. For this application, blots would be coated with
albumin or gelatin, reacted with radiolabeled lectin (Subheading 3.2., steps 6–8), and
recoated with albumin or gelatin (Subheading 3.4., steps 2 and 3) prior to drying. After
autoradiography, the radiolabeled lectin would be solubilized in warm SDS under reducing
conditions (Subheading 3.3.)
Acknowledgments
The technical assistance of Sharon J. McLaughlin, Phyllis A. Svingen, and Timothy
J. Kottke in the preparation of Figs. 1–4, as well as secretarial assistance of Deb Strauss,
are gratefully acknowledged. Studies in my laboratory are supported by grants from
the National Cancer Institute.
References
1. Gershoni, J. M. and Palade, G. E. (1983) Protein blotting: principles and applications.
Analyt. Biochem. 131, 1–15.
2. Beisiegel, U. (1986) Protein blotting. Electrophoresis 7, 1–18.
3. Stott, D. I. (1989) Immunoblotting and dot blotting. J. Immunol. Meth. 119, 153–187.
4. Kaufmann, S. H. and Kellner, U. (1998) Erasure of Western blots after autoradiographic or
chemiluminescent detection, in Methods in Molecular Biology, Vol. 80, Humana Press,
Totowa, NJ, pp. 223–235.