Protein Protocols Protein Protocols

1032 Hammerl et al.
can “corrode” the plastic surface. As a consequence, the transparency of these parts may
be lost with time, which can cause difficulties in handling, (e.g., sample application).
3. While washing the gel surface, do not leave water on the gel for too long a time, as this
would dilute the buffer and SDS concentration in the upper part of the gel.
4. The protocol for SDS-PAGE described here uses the discontinuous buffer system
of Laemmli and colleagues (3). For the band sharpening effect of this system a sharp
increase in pH and buffer concentration between stacking and separating gel is essential.
As soon as the stacking gel is casted, however, the two different buffer systems start to
diffuse into each other, and consequently interfere with the beneficial effect of the system.
Therefore, it is essential to minimize the time between casting the stacking gel and starting
the electrophoresis. For this reason, we do not recommend casting the stacking gels
before the samples are ready for application. The polymerization of the stacking gel is
complete as soon as a second fluid phase is visible between the gel and the butanol phase.
5. For repeated runs, the electrophoresis buffer may be reused several times. After each run,
mix the cathodal and the anodal buffers to restore the pH value. Reuse this buffer only as
the anodal buffer in the following runs. For the cathode, use fresh buffer in every run.
Reused buffer contains chloride ions from the separating gel of the previous run, which
would eliminate the effect of the discontinuous buffer system.
6. Other blotting systems (e.g., in a tank blot module in 10 mM Na 2CO 3 or other buffer
systems) are also suitable for this purpose. From our experience, the best results are
obtained by semidry blotting, especially with respect to homogeneous transfer efficiency.
7. The working dilution of antiserum depends on parameters such as antibody titer and affinity,
and therefore varies with the material used. For this reason, no suggestions on antiserum
dilution and time of incubation are given in this protocol. It should be adjusted
according to the experience of the investigator with the material at hand. However, conditions
that ensure a maximum of specificity are a crucial requirement, especially for the
cross-blotting method. Therefore, do not use too high a concentration of antiserum and
add BSA at 1 mg/mL to minimize unspecific protein–protein interactions. If problems
occur, increasing the ionic strength to 0.5 M NaCl may be helpful.
8. A rigorous washing procedure was included in the protocol to optimize the washing efficiency.
Possibly, this is not necessary for all applications, depending on the materials
used. Keep in mind that any antibody, which remains unspecifically attached to the donor
blot, will increase background and unspecific signals on the receptor blot.
9. Make sure that the donor and receptor blot are cut of exactly equal size and placed onto
each other accurately. This will facilitate the identification of individual spots in the final
result. It may be helpful to mark the position of two NC sheets by penetrating them with a
needle at three sites after they have been placed together.
10. The relatively long electrophoresis time used in the cross-blot step is intended to maximize
the efficiency of antibody elution from the donor blot. However, some antibody
species may not survive such a long exposure to 1 M NaSCN. This could be indicated by
increased unspecific signals or low sensitivity in the final results. In such a case, try a
shorter electrophoresis time or a lower concentration of NaSCN in the cathodal Whatman
stack. Generally, it may be advantageous to minimize the electrophoresis time in this step
so as not to exceed the buffer capacity in the Whatman stacks. This would cause a change
in pH, which might affect the direction of antibody migration.
11. This is the most crucial step of the entire procedure. Right after the electrotransfer, the
antibodies are not yet bound to the antigens on the receptor blot due to the presence of
NaSCN. Instead, they are most likely trapped in the pores of the NC sheet and loosely
attached on the surface of the dialysis membrane. Therefore, any movement of the recep-