Protein Protocols Protein Protocols

Reutilization of Western Blots 449
signals only when used at a dilution of 1:5 or 1:10. When attempting to blot with an
antiserum for the first time, it is reasonable to try one or more arbitrary concentrations in
the range of 1:10 to 1:500. If a strong signal is obtained at 1:500, further dilutions can be
performed in subsequent experiments.
8. Different investigators incubate blots with primary antibodies for different lengths of time
(reviewed in ref. 3). Preliminary studies with some of our reagents have indicated that
the signal intensity is greater when blots are incubated with antibody overnight rather than
1–2 h at room temperature.
9. Most diluted antibody solutions can be reused multiple times. They should be stored at
4°C after additional aliquots of penicillin/streptomycin and sodium azide have been added.
Some workers believe that the amount of nonspecific (background) staining on Western
blots diminishes as antibody solutions are reutilized. Antibody solutions are discarded or
supplemented with additional antibody when the intensity of the specific signal begins to
diminish.
10. Choice of wash buffer after incubation with primary antibody: 2 M urea is included in the
suggested wash buffer to diminish nonspecific binding. Alternatively, some investigators
include a mixture of SDS and nonionic detergent (e.g., 0.1% (w/v) SDS and 1% [w/v]
Triton X-100] in the wash buffers. For antibodies with low avidity (especially monoclonal
antibodies and anti-peptide antibodies), the inclusion of 2 M urea or SDS might diminish
the signal intensity. These agents are, therefore, optional depending upon the properties of
the primary antibody used for blotting.
11. It is important to avoid sodium azide when using horse radish peroxidase-coupled antibodies,
as the azide inhibits peroxidase (26). For this reason, adding milk to PBS immediately
before use of each aliquot of this buffer is advisable.
12. The concentration of secondary antibody is determined empirically. Most suppliers recommend
a dilution for their reagents, typically to the 0.1–0.5 µg/mL range.
13. The same ECL reagent can be transferred from one blot to another to develop several blots
simultaneously.
14. The length of exposure required to give a good signal varies depending on the abundance
of the antigen, the quality of the primary and secondary antibodies, and the nature of the
solid support utilized. One convenient way to proceed is to expose each blot for an arbitrary
time (e.g., 2 min) and then increase or decrease the exposure time based on the
results of the trial exposure.
15. The signal obtained with some chemiluminescence reagents decays with a halftime of
30–60 min. With these reagents, it is important to expose film promptly after treating blots.
Other chemiluminescence reagents continue to generate a strong signal for many hours.
4.3. Dissociation of Antibodies after Chemiluminescent Detection
16. Choice of erasure buffer: Experiments showing the effect of various erasure buffers on
removal of primary antibodies are illustrated in Fig. 2. After detection by enhanced
chemiluminescence, bound antibodies can be solubilized using 6 M guanidine hydrochloride,
8 M urea, or 2% SDS (middle panel, Fig. 2). None of these treatments elutes
significant amounts of antigen from nitrocellulose (lower panel, Fig. 2). After enhanced
chemiluminescent detection, antibodies appear to be more easily removed from PVDF
than from nitrocellulose (cf. lanes 6 and 13 in Fig. 2).
17a. Temperature of incubation: When blotting is performed after immobilization of polypeptides
on nitrocellulose, complete removal of antibodies by either 8 M urea or 2% SDS
requires heating to >50°C for 30 min (ref. 16; see also Fig. 2, lanes 4–7).
17b. Length of incubation. When blotting is performed on nitrocellulose, complete dissociation
of radiolabeled antibodies at 70°C requires a minimum of 20 min incubation with