MIPS activity assay

Cells expressing His-Xpress tagged MIPS were grown to the early stationary phase and lysed in buffer (50 mM Tris-HCl pH 7.5, 0.6 M sorbitol, 0.3 M NaCl, 1X protease inhibitor and 1X phosphatase inhibitor) by vortexing with acid-washed glass beads at 4°C. MIPS protein was purified from cell extracts using the PureProteome^™ Nickel Magnetic Bead System (Millipore). Purified MIPS protein was dialyzed (1 mM Tris acetate pH 8.0, 0.05 M dithiothreitol, 0.025X protease inhibitor and 0.1X phosphatase inhibitor) and concentrated with an Amicon Ultra-0.5 Centrifugal Filter (Millipore). MIPS protein concentration was determined by Bradford assay. Enzymatic activity of 3 μg purified MIPS was determined by enzyme-coupled colorimetric assay [44].