Protein Protocols Protein Protocols

Immunoblotting Using Secondary Ligands
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Recently, chemifluorescent detection systems have been introduced for both alkaline phosphatase
and horseradish peroxidase (see Chapter 56). These sytems are based on the
hydrolysis or oxidation of a fluorogenic substrate to yield an insoluble product that is
visualized by fluorimetry using a charge coupled device CCD camera or other imaging
system. The sensitivities of commercially developed systems such as FluoroBlot (Pierce
Chemical) and Vistra ECF (Amersham Pharmacia Biotech) are claimed to be comparable
to those of chemiluminescent systems. Relative to chemiluminescence, the potential benefits
of fluorescence detection are linearity of response over a greater range of signal,
adjustable sensitivity, and stability of signal. Because light emission is dependent on
exposure to the excitation light, a single fluorochrome molecule can be excited repeatedly,
and a fluorescent blot can be stored and rescanned after several weeks with little loss
of signal. In contrast, using chemiluminescence detection, light emission peaks within
minutes of substrate addition and decays within a few hours. Subsequent revisualization
requires reapplication of substrate and relies on the extent to which the activity of phosphatase
or peroxidase is retained during storage.
3. Iodination of protein A using Bolton and Hunter reagent (see Chapter 134) labels the
ε-NH 2 group of lysine, which apparently is not involved directly in the binding of protein
A to the Fc region of IgG. This method is preferable to others, such as those using chloramine
T or iodogen, which label tyrosine. The only tyrosine residues in protein A are associated
with Fc binding sites, and their iodination may reduce the affinity of protein A for
IgG (11).
4. Many workers include up to 0.05% sodium azide in the antibody and washing buffers to
prevent microbial contamination. However, azide inhibits horseradish peroxidase. Therefore,
do not use buffers containing azide when using this enzyme.
5. In the original description of this protocol 5-bromo-4-chloroindolyl phosphate was made
up as a stock solution in dimethylformamide. However, this is not necessary if the disodium
salt is used since this compound dissolves readily in aqueous buffers.
6. Urea peroxide may be used instead of hydrogen peroxide as a substrate for peroxidase.
The problems of instability, enzyme inactivation and possibility of caustic burns associated
with hydrogen peroxide are eliminated by using urea peroxide. A 10% (w/v) stock
solution of urea peroxide is stable for several months and is used at a final concentration
of 0.1% in the peroxidase substrate mixture.
7. If desired, the nitrocellulose filter may be stained with Ponceau S immediately after
electroblotting. This staining apparently does not affect the subsequent immunodetection
of polypeptides, if the filter is thoroughly destained using PBS before incubation with the
antiserum. In addition to confirming that the polypeptides have been transferred successfully
onto the filter, initial staining allows tracks from gels to be separated precisely and
probed individually. This is useful when screening several antisera.
8. Nonspecific binding is a common problem in immunoblotting. Several factors are important
in reducing the resulting background.
First, the filter is washed in the presence of an “inert” protein to block the unoccupied
binding sites. Bovine serum albumin is the most commonly used protein, but others, such
as fetal calf serum, hemoglobin, gelatin, and nonfat dried milk, have been used successfully.
Economically, the latter two alternatives are particularly attractive.
The quality of protein used for blocking is important, as minor contaminants may interfere
with either antigen–antibody interactions or the binding of protein A to IgG. These contaminants
may vary between preparations and can be sufficient to inhibit completely
the detection of specific polypeptides. Routinely we use bovine serum albumin (fraction V)
from Sigma–Aldrich (product no. A 4503), but no doubt albumin from other sources is