2 mg of cellular extract from Hek 293 cells were pre-cleared with 50 ?l of protein G sepharose for 30 minutes at 4 °C before the supernatant was incubated with 5 ?g of anti- MSK1 (26 – 44) and 50 ?l of protein G sepharose for 1 hour at 4 °C. The supernatant was removed and the pellets washed with lysis buffer before being subjected to SDS/PAGE and transferred to nitrocellulose membrane. The membrane was then immunoblotted with anti-GST-MSK1 (S804) at a concentration of 1 ?g/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).