Protein Protocols Protein Protocols

Immunoblotting Using Secondary Ligands
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by incubating the membrane with an appropriate chromogenic substrate that is converted
to a colored, insoluble product. The latter precipitates onto the membrane in the area of
enzyme activity, thus identifying the site of the antibody-antigen complex (see Note 2).
Both antigens and antisera can be screened efficiently by immunoblotting. Probing
of a crude extract after fractionation by SDS-PAGE can be used to assess the specificity
of an antiserum. The identity of the antigen can be confirmed using a complementary
technique, such as immunoprecipitation of enzyme activity. This information
is essential if the antibodies are to be used reliably. Once characterized, an antiserum
may be used to identify antigenically related proteins in other extracts
using the same technique (see Note 17). Examples of the potential of immunoblotting
have been described by Towbin and Gordon (7).
2. Materials
1. Electrophoretic blotting system, such as Trans-Blot, supplied by Bio-Rad.
2. Nitrocellulose paper: 0.45 µm pore size.
3. Protein A derivative.
a. Alkaline phosphatase-conjugated protein A obtained from Sigma–Aldrich Co. Dissolve
0.1 mg in 1 mL of 50% (v/v) glycerol in water. Store at –20°C.
b. Horseradish peroxidase-conjugated protein A obtained from Sigma–Aldrich Co. Dissolve
0.1 mg in 1 mL of 50% (v/v) glycerol in water. Store at –20°C.
c. 125I-labeled protein A, specific activity 30 m Ci/mg. Affinity-purified protein A, suitable
for blotting, is available commercially (see Note 3). 125I emits γ-radiation. Be
sure that you are familiar with local procedures for safe handling and disposal of
this radioisotope.
4. Secondary antibody: A wide range of both alkaline phosphatase and horseradish
peroxidase conjugated antibodies are available commercially. They are usually supplied
as an aqueous solution containing protein stabilizers. The solution should be stored under
the conditions recommended by the supplier. Ensure that the enzyme-linked antibody is
against IgG of the species in which the primary antibody was raised.
5. Washing solutions: Phosphate-buffered saline (PBS): Make 2 L containing 10 mM
NaH2PO4, 150 mM NaCl adjusted to pH 7.2 using NaOH. This solution is stable and may
be stored at 4°C. It is susceptible to microbial contamination, however, and is usually
made as required.
The other washing solutions are made by dissolving the appropriate weight of bovine
serum albumin or Triton X-100 in PBS. Dissolve bovine serum albumin by rocking the
mixture gently in a large, sealed bottle to avoid excessive foaming. The “blocking” and
“antibody” solutions containing 8% albumin may be stored at –20°C and reused several
times. Microbial contamination can be limited by filter-sterilizing these solutions after
use or by adding 0.05% (w/v) NaN3 (but see Note 4). Other solutions are made as required
and discarded after use.
6. Alkaline phosphatase substrate mixture:
a. Diethanolamine buffer. Make up 100 mM diethanolamine and adjust to pH 9.8 using
HCl. This buffer is usually made up as required, but may be stored at 4°C if care is
taken to avoid microbial contamination.
b. 1 M MgCl2. This can be stored at 4°C.
Combine 200 µL of 1 M MgCl2, 5 mg of nitroblue tetrazolium, 2.5 mg of 5-bromo-4chloroindolyl
phosphate (disodium salt; see Note 5). Adjust the volume to 50 mL using
100 mM diethanolamine buffer. Make up this reaction mixture as required and protect
from the light before use.