Protein Protocols Protein Protocols

Western Cross-Blotting 1033
tor sheet against the anodal surface of the dialysis membrane will result in an extreme
decrease of the spot sharpness. The donor blot is removed in this step so that it does not
compete with the antigens on the receptor blot during the subsequent reequilibration step.
Given a sufficient sensitivity according to our experience with previous experiments, the
removal of the donor blot may be omitted.
12. Alternate staining procedures are also possible, for example, second antibody conjugated
to alkaline phosphatase and staining with nitroblue tetrazolium (NBT) and bromochloroindolyl
phosphate (BCIP).
As the staining intensity on the receptor blot is significantly lower than that on the reference
strips, one may wish to employ a more sensitive detection system such as luminogenic
enzyme substrates. However, some of these systems require the use of special membranes.
As of now we have no experience to indicate that these membranes are also compatible
with the cross blot procedure.
13. Interpretation of results: Only positive signals should be taken as a result. The failure of a
band on the donor blot to react with a band on the receptor blot does not necessarily prove
the absence of cross-reactivity. Such negative results could also be due to inefficient elution
of an antibody from the donor blot or to denaturation of the eluted antibody. Likewise,
the concentration of the antigen and/or antibody on the donor blot may be too low to
give a signal on the receptor blot. As a rule, do not necessarily expect a signal on the crossblot
from donor blot bands which give only faint signals on the donor blot reference strips.
Generally, it is sometimes difficult to judge the specificity of an antigen–antibody
reaction. This is more likely for antibodies, which have been exposed to low pH values or
chaotropic agents. With cross-blot results, be doubtful about donor blot bands, which give
a signal with each band on the receptor blot. Also, receptor blot bands that react with each
donor blot band should generally not be taken too seriously, unless special circumstances
let you expect such a behavior.
When working with different antigen mixtures on donor and receptor blots, respectively,
include a homologous cross-blot as a control and reference. This is done by crossblotting
antibodies from a donor blot to a receptor blot containing the same antigens as the
donor blot. Such homologous cross-blots have a “natural” internal reference because each
antigen band on the donor blot is crossing “itself” on the receptor blot. In this way, the
cross-blot yields a diagonal line of spots that are helpful in estimating both the sensitivity
and the specificity you can expect with a particular band on the donor blot. Similarly, a
homologous cross-blot may also be carried out with the receptor antigens. This will help
to estimate the resistance of the receptor antigens against exposure to the cross-blot conditions.
References
1. Hammerl, P., Hartl, A., and Thalhamer, J. (1992) A method for the detection of serologically
crossreacting antigens both within and between protein mixtures: the Western cross
blot. J. Immunol. Meth. 151, 299–306.
2. Beall, J. A. and Mitchell, J. F. (1986) Identification of a particular antigen from a parasite
cDNA library using antibodies affinity purified from selected portions of Western blots.
J. Immunol. Meth. 86, 217–223.
3. Laemmli, E. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680–685.