Detection of pH-dependent FcRn binding of IgG in rabbit yolk sac samples.

<p>In this assay we analyzed the pH dependent binding of the lysate of rabbit yolk sac of 24 days post coitum (dpc) embryos at pH 6.0 and pH 7.4 to rabbit IgG that was coupled to a Sepharose matrix. Absorbed proteins were then eluated from the matrix and the presence of the FcRn was Western blot tested in these samples as well as in the unbound fractions. The eluted samples contain FcRn only if binding occurred at pH 6 but not at pH 7.4 (bands 5 and 6, respectively). Confirming this result, we could not detect FcRn in the unbound fraction when binding occurred at pH 6.0 (i.e. all FcRn molecule bound to matrix) but FcRn remained in the unbound fraction when the pH of reaction media was neutral (i.e. at this pH no FcRn bound to the IgG-matrix) (bands 3 and 4, respectively). Recombinant bovine FcRn <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028869#pone.0028869-Kacskovics4" target="_blank">[46]</a> and rabbit yolk sac protein extract were used as positive controls (bands 1 and 2, respectively).