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Johns Hopkins University School of Medicine
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AS 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.

 

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