1. Resuspend cells (vegetative or developed) in phosphate buffer at 1 x 10 8
2. Add 50 ul cells to 50 ul 20 nM 3H-cAMP in 5-10 mM DTT (for background
binding, add same amount of hot cAMP plus 10 uM cold cAMP) in 1.5 ml eppendorf tube.
Incubate 30 sec to 3 min.
3. Add 1.2 ml AS.
Also add BSA to 1 mg/ml. Mix by brief vortexing. Incubate on ice for
4. Spin at full speed with cold room microfuge for 1-2
mins. Aspirate out supernatant, wash with 1.2 ml cold AS. Spin down cell pellet again with cold-room
microfuge. Aspirate out supernatant.
in 100-150 μl 1% SDS or 0.1% formic acid. Transfer
into counting vials and add 3 ml scintillation fluid. Count.
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