Fig. 7. (A) Expression and purification of full-length recombinant human and mouse usherin protein. The full-length cDNAs for human and mouse usherin were cloned into pcDNA 3.1 and transfected into 293 cells. Clones secreting the highest amounts were grown, the supernatant concentrated and recombinant usherin purified by affinity chromatography over nickel columns. Samples of the purified protein were subjected to SDS-PAGE, and the protein stained with Coomassie Blue. The arrow indicates stained protein of the predicted molecular size for usherin (175 kDa). (B) Competitive inhibition of immunostaining using recombinant usherin protein. (Top) Immunostaining with antibody 2 (see Fig. 1). (Bottom) The antibody was mixed with 100 ng of recombinant usherin protein prior to applying it on the tissue section. Absence of staining in B proves that the recombinant protein shown in A is indeed usherin. Both mouse and human usherin competed equally well.