Protein Protocols Protein Protocols

448 Kaufmann
Fig. 3. Conditions for dissociating radiolabeled antibodies after immobilization of polypeptides
on various solid supports. Replicate samples containing 2 × 10 6 rat liver nuclei were
subjected to SDS-PAGE followed by transfer to nitrocellulose, PVDF, or Nytran nylon sheets
as indicated (see Note 2). After staining with fast green FCF and treatment with TSM buffer to
coat unoccupied protein binding sites (Subheading 3.1., steps 8–11), blots were incubated
with chicken polyclonal antiserum that reacts with the nuclear envelope polypeptide lamin B 1
(25) followed by 125 I-labeled rabbit anti-chicken IgG (Subheading 3.4., step 1). Half of each
blot (lanes 6–10) was recoated with milk-containing buffer for 5 min prior to drying
(Subheading 3.4., step 2) and the other half (lanes 1–5) was dried without being recoated with
protein. After autoradiography confirmed that the signals in all lanes of a given panel were
comparable (upper panels), samples were incubated for 30 min at the indicated temperature
with 50–62.5 mM Tris-HCl (pH 6.8) containing 100 mM 2-mercaptoethanol and one of the
following denaturing agents: 2% (w/v) SDS, 6 M guanidine hydrochloride (GuHCl) or 8 M
urea. Following this erasure procedure, strips were subjected to autoradiography again (bottom
panels). In each case, untreated strips (lanes 1 and 6) served as controls. This analysis leads to
several conclusions. First, recoating with milk prior to drying (Subheading 3.4., step 2) does
not affect the amount of radiolabeled antibody initially bound to the blots (cf. lanes 1 and 6).
Second, the efficacy of various treatments at removing these antibodies varies depending upon
the solid support. For nitrocellulose or PVDF, coating of the blots with protein prior to drying
facilitates the subsequent dissociation of the anti-lamin B antibodies. In both cases, SDS-containing
buffer (lanes 7 and 8) is more effective than guanidine hydrochloride (lane 9) or urea
(lane 10). For nylon, SDS is again slightly more effective than urea at dissociating the antibodies
(cf. lanes 2, 5, and 7, 10). Interestingly, when SDS-containing erasure buffer is used, it is
not necessary to recoat nylon with protein prior to drying for autoradiography (cf. lanes 2 and 7).
This is in contrast to nitrocellulose or PVDF, which were examined in the same experiment.