Protein Protocols Protein Protocols

442 Kaufmann
Fig.1. Reutilization of immunoblots without intervening dissociation of antibodies. After
staining with fast green FCF and coating with TSM (Subheading 3.1., steps 8–11), a single
strip containing total cellular protein from 3 × 10 5 K562 human leukemia cells was sequentially
subjected to immunoblotting using the following primary (secondary) reagents:
chicken anti–B23 (peroxidase-coupled goat anti-chicken IgG), rabbit anti-protein kinase Cδ
(peroxidase-coupled goat anti-rabbit IgG), mouse anti-poly(ADP-ribose) polymerase (peroxidase-coupled
goat anti-mouse IgG), and mouse anti-procaspase-9 (peroxidase-coupled goat
anti-mouse IgG). At each step, bound secondary antibodies were detected using ECL-enhanced
chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). At each step,
primary antibody was added in TSM buffer, which contains sodium azide. Note that the peroxidase
bound to the blot at each step is not catalytically active at subsequent steps (cf. lanes 1–3),
whereas the previously bound primary antibodies react with fresh secondary antibodies at
subsequent steps (cf. lanes 3 and 4). Numbers at left, molecular masses of the respective
polypeptides in kilodaltons.
blot. It is important to stress, however, that each of these probings involves a different
secondary antibody. When the same blot is subsequently probed with a second mouse
immunoglobulin G (IgG) monoclonal antibody, the previously bound mouse IgG is
detected by the peroxidase-coupled anti-mouse IgG (e.g., Fig. 1, lanes 3 and 4). Thus,
sequential probing works well when multiple primary antibodies raised in different
species are available or when antigens of different molecular weights can be unequivocally
detected using antibodies from the same species. This approach can become problematic,
however, if the antigens of interest have similar molecular weights or the
immunological reagents cross-react with multiple species of different molecular