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In a final reaction volume of 25 μL, human c-RAF (5-10 mU, Millipore) was incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.66 mg/mL myelin basic protein, 10 mM Mg acetate and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol). The reaction was initiated by the addition of the MgATP mix. After incubation for 40 min at room temperature, the reaction was stopped by the addition of 3% phosphoric acid solution. The reaction was then spotted onto a P30 filtermat and washed prior to drying and scintillation counting. All measurements were performed in duplicates.
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