Fig. 1. Distribution of 78 kDa and 80 kDa isoforms of CALK in wild-type, unactivated gametes. (A) Both isoforms of CALK are primarily localized in the cell bodies of unactivated wild-type gametes. Flagella (30 µg) and cell bodies (15 µg) isolated from unactivated wild-type gametes were analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (B) Absence of membrane-associated CALK in unactivated gametes. Unactivated wild-type gametes were separated into freely soluble, NP-40-soluble and NP-40-insoluble fractions and analysed by SDS-PAGE and immunoblotting with anti-CALK antibody. (C) Sucrose gradient analysis of CALK of unactivated gametes. NP-40 extracts of unactivated wild-type gametes were separated on 5-20% sucrose gradients and equal portions of each fraction were analysed by anti-CALK immunoblotting. (We used NP-40 to disrupt the gametes in this experiment for consistency with the experiments in Fig. 2.) All 18 fractions from the gradient were analysed by SDS-PAGE and immunoblotting but only the top 9, which contained all of the CALK detected on the gradient, are shown here.