The supernatant from the equilibrated beads was added and placed on a rotating mixer for 1 hour. Spinning was accomplished for 5 minutes at 700g, and the unbound supernatant discarded. The beads were then resuspended in denaturing buffer and spined for a further 5 minutes at 700g. The supernatant was discarded, but 100 Î¼l of it was retained for SDS-PAGE- Wash 1. Thereafter, steps 5 and 6 were repeated. Again, the supernatant was discarded, and another 100 Î¼l of retained for SDS-PAGE- Wash 2.
The beads were then resuspended in the same volumes of denaturing buffer, in addition to solubilising 20 mM imidazole as the beads. Spinning was accomplished for 5 minutes at 700g. The supernatant was discarded but 100 Î¼l of it was retained for SDS-PAGE- IMIDAZOLE WASH. The beads were resuspended in a similar buffer volume as that of the beads, with 100mM imidazole, and spinning accomplished for 1 minute at 700g. All the supernatants was retained.