Protein Protocols Protein Protocols

950 Diano and Le Bivic
Fig. 3. Second dimension with several IEF gels. Several IEF gels are cut, 0.5 cm above and
0.5 cm below the isoelectric point of the protein of interest. They are placed side by side at the
top of the second dimension slab gel. Thus, only one SDS gel is needed to collect several spots
of interest.
4. This is a crucial point in the procedure. If too much PBS is added, the pieces of nitrocellulose
will swirl around the probe and disintegration does not occur. In this case, the nitrocellulose
pieces should be allowed to settle to the bottom of the tube before sonication and
the excess buffer drawn off with a syringe or other suitable instrument (70–80 µL of PBS
is sufficient for about 0.4 cm 2 of nitrocellulose). For these quantities, one or two 10-s
cycles suffice to get powdered nitrocellulose. We mention the volume as a reference since
the surface of nitrocellulose-bound antigen may vary. In every case the volume of PBS
must be adjusted. We experienced that PVDF is more resistant to sonication than nitrocellulose
and thus several cycles of freezing and thawing are necessary.
5. What is an appropriate amount of antigenic protein to inject? There is no absolute answer
to this question. It depends both on the molecular weight of the protein and also on its
antigenicity. It is well known that if the amount of antigen is very low (0.5–1 µg for the
classic method with Freund’s adjuvant), there is no antibody production; if the amount of
antigen is very high (e.g., several hundred micrograms of highly antigenic protein), antibody
production might also be blocked.
It would appear that in our method, a lower amount of antigen is required for immunization;
the nitrocellulose and PVDF act as if they progressively release the bound protein,
and thus, the entire amount of protein is used progressively by the cellular machinery.
Our experiments show that a range of 10–40 µg for the first immunization generally
gives good results, although, in some cases, 5 µg of material is sufficient. The nitrocellulose
powder has the additional effect of triggering an inflammatory process at the sites of
injection, thus enhancing the immune response, as does Freund’s adjuvant by means of
the emulsion of the antigenic protein with the tubercular bacillus; macrophages abound in
the inflamed areas.
6. It is perhaps worth noting that careful attention should be paid to the condition of the
rabbit at time of bleeding. We bleed the rabbits at the lateral ear artery. When the rabbit is
calm, 80–100 mL of blood may be taken. The best time for bleeding is early in the morning,
and after the rabbit has drunk. Under these conditions, the serum is very clear. It is
essential to operate in a quiet atmosphere. If the rabbit is nervous or under stress, the
arteries are constricted so strongly that only a few drops of blood can be obtained. Note
that to avoid clotting, the needle is impregnated with a sterile sodium citrate solution by
drawing the solution into the syringe 3×. The atmosphere in the animal room should be
also stressless for the rats to obtain enough blood.