Ammonium Sulfate Binding Assay for Cells

1. Resuspend cells (vegetative or developed) in phosphate buffer at 1 x 10 8 /ml .

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 >3 mins.

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.

5. Resuspend in 100-150 μl 1% SDS or 0.1% formic acid. Transfer into counting vials and add 3 ml scintillation fluid. Count.