Protein Protocols Protein Protocols

412 Kruger
equally effective. The suitability of individual batches of protein should be checked using
antisera known to react well on immunoblots.
Second, the background may be reduced further by including nonionic detergents in the
appropriate solutions. These presumably decrease the hydrophobic interactions between
antibodies and the nitrocellulose filter. Tween 20, Triton X-100, and Nonidet P-40 at
concentrations of 0.1–1.0% have been used. In my experience, such detergents may
supplement the blocking agents described previously, but cannot substitute for these
proteins. In addition, these detergents sometimes remove proteins from nitrocellulose
(see Note 9).
Third, the nitrocellulose must be washed effectively to limit nonspecific binding. For this,
the volumes of the washing solutions should be sufficient to flow gently over the surface
of the filter during shaking. The method described in this chapter is suitable for 12 × 7 cm
filters incubated in 14 × 9 cm trays. If the size of the filter is significantly different, the
volumes of the washing solutions should be adjusted accordingly.
Finally, decreasing the incubation temperature to 4°C may greatly decrease the extent of
nonspecific background binding (12).
9. Protein desorption from the membrane during the blocking step and subsequent incubations
can result in the loss of antigen and decrease the sensitivity of detection (13,14). In
some instances, this problem may be reduced by incubating the membrane in 0.1 M phosphate
buffer, pH 2.0 for 30 min and then rinsing in PBS prior to treatment with the blocking
agent. Such acid treatment is particularly effective when using non-denaturing gel
blots, or SDS-PAGE blots transferred onto polyvinylidene difluoride rather than nitrocellulose
membrane (14).
Alternatively, polyvinyl alcohol may be used as a blocking agent (15). In comparative
tests, PBS containing 1 mg/mL polyvinyl alcohol produced lower background staining
than other commonly used blocking agents. Moreover, the blocking effect of polyvinyl
alcohol is virtually instantaneous, allowing the incubation time to be reduced to 1 min and
decreasing the opportunity for loss of protein from the membrane (15).
10. The exact amount of antibody to use will depend largely on its titer. Generally it is better
to begin by using a small amount of antiserum. Excessive quantities of serum tend to
increase the background rather than improve the sensitivity of the technique. Nonspecific
binding can often be reduced by decreasing the amount of antibody used to probe the
filter. Also, see Notes 15 and 16.
11. Deciding which form of detection system to use is largely a personal choice. Detection
using 125 I-labeled protein A is very sensitive. However, many researchers prefer to use
nonradioactive methods, and the sensitivities of chemiluminescent and chemifluorescent
systems approach that of radiolabeling. Comparison between the two enzymic detection
systems is difficult because the reported sensitivity limits of both systems vary considerably,
and most studies use different antigens, different primary antibodies, and different protocols.
Despite these uncertainties, alkaline phosphatase is generally considered more sensitive
than horseradish peroxidase when visualized using the standard colorimetric detection
systems described in this chapter. For routine work I prefer to use alkaline phosphatase
conjugated protein A.
12. The products of the peroxidase reaction are susceptible to photobleaching and fading.
Consequently, the developed filters should be stored in the dark, and the results photographed
as soon as possible. The products of the phosphatase reaction are reportedly stable
in the light. However, I treat such filters in the same way—just in case!
13. To increase sensitivity of the diaminobenzidine-based staining protocol replace the standard
substrate mixture with the following intensifying solution (16). Dissolve 100 mg of
diaminobenzidine in 100 mL of 200 mM phosphate buffer, pH 7.3. To this solution add,