GTPγSinduced Inhibition of Binding

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 Cellsa. Wash cells with DB and resuspend them to 2 X 10⁷ / mlb. Shake cells at 120 rpm for 1 hourc. Pulse cells with 100 nM cAMP for 4 hoursd. Basolate cells with 2 mM caffeine for 30 minFollowing steps are carried out at 4 ⁰Ce. Wash cells with DB and resuspend at 5 X 10⁷ / ml.

3. Assay

Filter-lyse cellsSpin 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 tubesMix 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 downAdd 100 ml 0.1% SDS, mix.count