| Assay Method Information | |
| | Drosophilia melanogaster kinase assay |
| Description: | D. melanogaster Polo kinase assays were performed as previously described40, using baculovirus-expressed Polo kinase. Enzyme was immunoprecipitated using antibody to mouse Polo (M294) and protein A beads that were incubated with increasing concentrations of 1 or 10 for 20 min. 5 ul of 1 ug ml 1 of dephosphorylated casein was added to an equal volume of 2 x kinase buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl2, 2 mM DTT and 1 mM EGTA). To this mixture was added 5 ul of protein A beads with immunoprecipitated Polo and 1 ul [γ-32P]ATP (10 mCi ml 1, Amersham), and 1.0 ul of 2 mM ATP. The mixture was incubated at 20 °C for 20 min. The phosphorylated substrates were identified by autoradiography after SDS-PAGE. |
| Affinity data for this assay | |
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