Figure 7. Identification of the sites on MBP phosphorylated by ERK8.

Bovine MBP (0.33 mg/ml) was phosphorylated by incubation for 10 min at 30 °C with Mg[γ-³²P]ATP (10⁶ c.p.m./nmol) and ERK8 (2 units/ml). Reactions were denatured in lauryl dodecyl sulphate and subjected to SDS/PAGE. After staining with Coomassie Blue, the MBP was excised from the gel, digested with trypsin and the resulting peptides were chromatographed on a Vydac C₁₈ column equilibrated in 0.1% (v/v) trifluoroacetic acid (A). The acetonitrile gradient is indicated by the diagonal line. Peptides T1 (B), T2 (C) from A were subjected to Edman degradation to determine the amino acid sequence, and to solid phase sequencing to identify the sites of phosphorylation, as in [24].