Basic Assay Protocol

From Assay Guidance Wiki

Whole membrane assay using WGA SPA beads

1. Incubate membranes, GTPγ35S, and compounds tested in 200 μl/well at room temperature for 30 – 60 minutes
2. Add 50 μl per well of suspended WGA beads (1 mg/well)
3. Seal plates and incubate for one hour or more at room temperature
4. Centrifuge at 200 x g and count plates in a Wallac microbeta

Antibody Capture assay

1. Incubate membranes as for whole membrane assay
2. Add 20 μl per well of 3% NP40 and incubate for 15 minutes
3. Add 20 μl per well of primary antibody and incubate for 15 minutes
4. Add 50 μl per well of anti-rabbit or anti-mouse SPA beads (1 mg)
5. Seal and incubate for three hours, centrifuge as above and count