Protein Protocols Protein Protocols

Immunoblotting of 2-DE Separated Proteins 223
tion is then carried out by comparing the signals of both matrices. Similarly, a fast and
simple method to produce print-quality like Ponceau replicas from blots was recently
described (63). The positive replicas are the same size as the blots and can be stored
without loss of intensity. This makes them useful for localizing immunoreactive spots in
complex 2-D electrophoretograms.
11. For a 16 × 18-cm membrane, we use 50 mL of solution in each washing and incubation
step. Volumes can be proportionally adjusted to other membrane dimension. It is important
that the membrane is entirely soaked in solution during the washing and incubation step.
We perform all steps of immunodetection in a flat glass vessel. Rotating glass cylinders
are also convenient. Do not incubate membrane in sealed plastic envelopes, because it
results in a very high background in ECL.
12. Our blocking procedure is suitable for routine use. However, special conditions and reagents
are required for immunoblotting with some antibodies, such as antiphosphotyrosine
antibodies (16,64,65). In this case, the presence of phosphorylated proteins in the blocking
solution could determine high immunochemical background staining. Information on
different blocking conditions can be found in the refs. 66–69. Chemicon have developed
a new blocking agent, composed of nonanimal proteins that ensure uniform blocking,
without nonspecific binding, eliminating all cross-reactivity with animal source antigens,
primary antibodies and secondary antibodies. Blocking with a nonionic detergent such as
Tween 20, without added protein, has also been used with the advantage that after
immunodetection the blot can be stained for total protein pattern (58,59,70) (see Note 10).
On the other hand, it has been found that blocking with detergent alone may cause loss of
transblotted proteins (71,72). Using PVDF membranes it is possible to employ a nonblock
technique: this method involves three cycles of methanol–water hydration of the membrane,
allowing multiple erasure and probing of the same blot with little or no loss of
signal (73).
13. Optimal dilution of the primary and secondary antibody should be determined by
immunoblotting of one-dimensional gels or dot blot analysis can be used. Working solutions
of antibodies can be stored at –20°C and used several times (74).
14. Secondary antibodies may often give problems of cross-reactivity, especially in the analysis
of samples containing antibodies (such as immunoprecipitates, immune tissue, plasma,
etc.), even when antibodies from different species are used. Bhatt et al. proposed a method
to avoid this problem, suppressing extraneous signals in immunoblots. This method consist
in preconjugating the primary with the secondary antibody before incubation. By this
procedure, signals from secondary antibodies are completely quenched (75).
15. ECL detects horseradish peroxidase conjugated antibodies through oxidation of luminol
in the presence of hydrogen peroxide and a phenolic enhancer under alkaline conditions.
ECL reagents are capable of detecting 1–10 pg of protein antigen. Alternative enhancers
that extend the duration of light emission and enable detection of even smaller quantities
of target protein (in the order of femtograms) have recently been developed. One such
system is ECL plus (Amersham Pharmacia Biotech) (76). These systems are suitable for
use with charge-coupled device (CCD) camera that require longer exposure times for good
quantification of immunoreaction.
16. In case chemiluminescence is too strong or background is too high, one can switch to
detection with a chromogenic substrate.
We use 4-chloro-1-naphthol (77) as chromogenic substrate, according to the following
protocol:
a. After ECL detection (or after step 7 of Subheading 3.3.1.), wash the membrane briefly
with 0.05 M Tris-HCl, pH 6.8.
b. Soak it in developing solution (20 mL of 0.05 M Tris-HCl, pH 6.8; 7 µL of H 2O 2 30%
(v/v); 5 mL 4-Chloro-1-naphthol 0.3% (w/v) in methanol) until the color appears.