Fig 3.
Measurement of GST-L–His-Ran affinity by SPR. (A) Representative SPR sensorgram curves for His-Ran (5 μg/ml, flowed over both GST and GST-L surfaces), RCC1-GST (0.5 μg/ml), and combined binding to GST-L (initially, 10 μl/min, 75 μg total, 25°C), using amine-coupled monoclonal antibody-GST surfaces on CM5 chips (GE Healthcare) and a BIAcore 2000 instrument. Only His-Ran in the presence of RCC1-GST over a GST-L chip reached equilibrium binding when measured over 20 min (1,200 s). At this point, the buffer was switched to one without protein (for 300 s), and monitoring was continued. (B) His-Ran binding to the GST-L surface in the presence of RCC1-GST is shown as normalized values, after taking into account all nonspecific binding controls (i.e., RCC1-GST and antibody only). Triplicate SPR determinations like this, with and without added GDP/GTP (10 μM), gave similar processed curves and were used for the KD calculations whose results are shown in Table 1.