Rat tissue extracts (40 μg of protein) and HEK 293 cellular extract (40 μg of protein) were subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-WNK1 at 1 μg/ml. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).
WNK1 and pre immune IgG antibodies were covalently coupled to protein G Sepharose in a ratio of 1 mg of antibody to 1 ml of resin using a dimethyl pimelimidate cross- linking procedure. As a pre-clearing step, 50 mg of rat testis lysate was incubated at 4 °C for 20 mins on a rolling shaker with 0.5 ml of protein G-Sepharose. The supernatant was then incubated at 4 °C for 1 hour on a rolling shaker with 50 μl of WNK1 or IgG-Protein G-Sepharose conjugated antibodies. The immunoprecipitates were washed four times with 10 ml of 10 mM Tris-HCl pH 8 / 0.1 mM EGTA. The resin was resuspended in SDS-Page sample buffer and subjected to SDS-Page analysis with staining with Coomassie blue.