Temperature-dependent kinetic measurements of the E3 ligase (RanBP2). Assay conditions are described in Basic Protocol 7. (A) Immunoblots used to detect GST-Sp100-SUMO conjugation at four Sp100 concentrations (2.1, 4.2, 4.6 and 8.4 μM) and at different temperatures (25, 28, 31, and 373C). (B) Plots of [E]/Vo versus 1/[GST-Sp100] based on data from (A) are linear which is indicates that the conditions are appropriate for Michaelis-Menten kinetics. The Km/kcat ratios are measured from the slope of the double-reciprocal plots (Lineweaver-Burk), and correspond to the net transfer rate constant for SUMO transfer from E2 to substrates.