Protein Protocols Protein Protocols

216 Magi et al.
Protein targets can be identified among thousands of non-modified proteins, by
means of antibodies specific for epitopes generated by post-translational modification
such as phosphotyrosine (15,16), carbonyl groups (17), nitrotyrosine (18), and covalent
adducts with drugs (19). Separation of whole electrophoretic patterns into subsets
of immunologically related spots may be a useful strategy for proteome research, as the
high sensitivity of immunodetection can extend the analysis to low copy number proteins,
because the sensitivity of immunostaining is often greater than that of silver staining
(12). The extent of modification can be assessed by the number and pI shift of
immunoreactive spots in the series, if the net charge of the protein is affected (20).
A modification of the immunoblotting procedures, called far-Western analysis, was
developed to detect different kinds of protein–protein interaction (21–23) and is now
also used for proteins separated by two-dimensional electrophoresis (24–26). The protocol
is similar to a western blot, except that a labelled protein is used as a probe to
detect specific protein–protein interactions. The probe can be directly biotinylated or
labelled with 32 P, 125 I or 35 S, or can be indirectly detected by a labelled antibody to the
protein used as the probe. Studies of protein–protein interaction can be useful to link a
protein with a specific function in known cellular processes (27).
2-D immunoblotting maintains the traditional steps consisting in:
1. Electrotransfer of proteins from polyacrylamide gel onto chemically resilient membrane.
2. Chemical staining of protein pattern on the transblotted membrane.
3. Saturation of the membrane.
4. Application of the primary, and then of the secondary, labeled antibody.
5. Detection of immunoreactive proteins by the label on the secondary antibody, for example,
radioactivity, enzyme activity.
In all steps, procedures developed for one-dimensional immunoblotting maintain
their significance and utility. However, matching immunoreactive spots to silverstained
spots in complex, high-resolution 2-DE patterns has become a far more difficult
task than matching bands in one-dimensional separations. As a consequence,
chemical staining of total protein pattern on the transblotted nitrocellulose or on equivalent
membrane plays a crucial role in 2-D immunoaffinity identification. Matching
itself is better carried out on digitized images with adequate computer and software
than by physically superimposing the chemiluminescent film or the membrane to the
silver-stained gel (cf. Subheading 3.5.).
The cost of reagents per sample is also higher than with the one-dimensional procedure,
as all volumes and surfaces are larger. As a preliminary step, it is convenient to
find out antibody working dilution by one-dimensional immunoblotting. In the same
way one can assess the need for a blank incubation with secondary antibody, omitting
the primary antibody. Costs for 2-D gel run and electrotransfer reagents can be also
reduced by reusing the electroblotted membrane consecutively with different antibodies
(cf. Subheading 3.4.). All reagents can be saved if “multiple immuno-2-D blotting,”
that is, simultaneous application of several primary antibodies, can be performed.
In this procedure, unambiguous immunodetection is allowed by different 2-D electrophoretic
parameters of proteins (12).