Sheep anti mouse dynabeads

Protein lysates from bloodstream form parasites were prepared as described (Das et al., 1998 (link)) except that the stated proteases and phosphatase inhibitors were replaced with cOmplete, Mini EDTA-free Protease inhibitor (Roche) and PhosSTOP (Roche). HA-tagged AEK1 from 1.8×10⁸ cells were immunoprecipitated as described (Park et al., 2002 (link)) using 2 μg mouse anti-HA clone16B12 (BioLegend) and 40 μl sheep anti-mouse Dynabeads (Invitrogen). For kinase assays, beads were washed once in kinase buffer (40mM Tris pH 7.5, 20mM MgCl₂, 0.1mg/ml BSA). Kinase assays were performed as described (Park et al., 2002 (link)) using 32P-γ-ATP and dephosphorylated myelin basic protein (MBP, MilliporeSigma) except the kinase buffer was replaced with the same buffer described above. One third of each reaction was resolved by SDS-PAGE. Gels were stained with Coomassie, fixed, dried, and labeled proteins detected by phosphorimaging. Signals were quantified using image J (Schneider et al., 2012 (link)). To normalize the amount of immunoprecipitated HA-tagged protein one quarter of each reaction was analyzed by western analysis with blots probed with rat anti-HA antibodies.