GTPγS-induced Inhibition of Binding

1. Chemicals and Equipment

DB Buffer, PB, cAMP, cAMP [2, 8- 3H] (25 Ci/mmol, 1.0 mCi/ml, 32, 000 nM), 10 mM GTPγS, Wacker silicone AR-20, Reagiergefab test tubes (Sarstedt), millipore 5 micrometer filter, 0.1M DTT.

2. Prepare Cells

a. Wash cells with DB and resuspend them to 2 X 10⁷ / ml
b. Shake cells at 120 rpm for 1 hour
c. Pulse cells with 100 nM cAMP for 4 hours
d. Basolate cells with 2 mM caffeine for 30 min
Following steps are carried out at 4 ⁰C
e. Wash cells with DB and resuspend at 5 X 10⁷ / ml.

3. Assay

Filter-lyse cells
Spin 10K, 5 min and resuspend membranes in PB at 10⁸ / ml equivalent.

Prepare reaction mixture plus and minus GTPγS
[3H] cAMP (2nM) 10 mM GTPgS M 0.1 M DTT 10 mM cAMP PB

Aliquot 200 ml silicone AR20 to the Sarstedt tubes
Mix 150 ml reaction mixture with 150 ml membrane, incubate in ice for 5 min, and then transfer 200 ml on the top of silicone oil.
Spin for 10 min.
Freeze on dry ice.
Clip the bottom off, spin upside down
Add 100 ml 0.1% SDS, mix.
count