Protein Protocols Protein Protocols

332 Gravel
PVDF membranes are more expensive but have high mechanical strength, high protein
binding capacity, and are compatible with most commonly used protein stains and immunochemical
detection systems. The chemical structure of the membrane offers excellent
resistance to acidic and organic solvents. This makes PVDF membrane an appropriate
support for N-terminal protein sequencing and amino acid composition analysis. Another
interesting advantage of PVDF matrix over the nitrocellulose is the possibility to visualize
the protein pattern on the blot without staining. After blotting, the PVDF membrane should
be placed on top of a vessel containing distilled water. The immersion of the membrane in
water should be avoided. It should be laid down at the surface. The protein spots (or
bands) contrast with the remainder of the membrane and can be visualized. To obtain a
clear image of the protein pattern, the surface of the membrane should be observed from
different angles and under appropriate lighting. This procedure, which was found unintentionally,
is easy to perform and allows rapid and good evaluation of the transfer quality.
Table 2 summarizes the most common matrices that can be used for transferring proteins
from polyacrylamide gels.
5. Whatever the membrane used, exceeding its binding capacity tends to reduce the signal
eventually obtained on blots. It can be assumed that excess protein, weakly associated
with the membrane, may be readily accessible to react with the probe in solution, but the
probe–protein complexes formed may then be easily washed off during the further processing
of the membrane (5). This situation does not occur if the proteins are initially in
good contact with the membrane.
For 2-D PAGE, the best recovery and resolution of proteins are obtained when loading
120 µg of human plasma or platelet proteins. When 400 µg of protein are separated by 2-D
PAGE and transferred on membrane, the spots are diffused and the basic proteins poorly
transferred (not shown). This could be attributed to overloading which prevents a good
separation of proteins and an adequate binding to transfer matrix.
6. It is very difficult to form large stacks of gel–membrane pairs. Even two pairs are often
associated with the introduction of air bubbles. We prefer to use a semidry unit to transfer
proteins from a single gel only.
7. Air bubbles create points of high resistance and this results in spot (or band) areas of low
efficacy transfer and spot (or band) distortion.
8. The quality or extent of the blocking step determines the level of background interference.
It has been recognized that the blocking step may also promote renaturation of epitopes
(23). This latter aspect is particularly important when working with monoclonal antibodies
(which often fail to recognize the corresponding antigenic site after electroblotting). Hauri
and Bucher (23) suggest that monoclonal antibodies may have individual blocking
requirements, probably due to different degrees of epitope renaturation and/or accessibility
of antibody under the various blocking conditions. Some common blocking agents are
listed in Table 3.
For immunoblotting on nitrocellulose membrane we obtain good results by using a
blocking solution made of 0.5% (w/v) BSA, 0.2% (w/v) Tween 20 and 5% (w/v) nonfat
dried milk in PBS buffer (137 mM NaCl, 27 mM KCl, 10 mM Na2HPO 4, 1.8 mM KH 2PO 4,
pH 7.4) (29). For lectin blotting on PVDF membrane, we use 0.5% (w/v) Tween 20 in
PBS buffer (8). (See also Chapter 106).
9. We store the blots at 4°C in PBS containing 0.005% (w/v) sodium azide. To evaluate the
effect of storage on blotted proteins, we stained with colloidal gold a nitrocellulose blot of
platelet proteins stored for a period of 4 mo in PBS–azide at 4°C. We observed a protein
pattern identical to the same blot stained immediately after western blotting (not shown)
On the other hand, when we dried a nitrocellulose blot at room temperature and stained