In Vitro Adenylyl Cyclase Assay

1. Starve cells 5 hours at 2 x 10⁷ cells/ml , pulsing with 50 nM cAMP every 6 min for 5 hour.

2. Plate 1 x 10⁷ cells on a 35 mm DB plate.

3. Spin at 1500 rpm and resuspend in PB at 1 x 10⁷ cells/ml.

4. Add caffeine (2 mM) and shake at 250 rpm for 30 min.

5. Spin at 1500 rpm at 4°C and wash twice in ice cold PM.

6. Resuspend at 8 x 10⁷ cells/ml in ice cold PM. Keep on ice. Take small aliquots for protein assay and Western analysis.

7. Lyse with an equal volume of Lysis Buffer through 5 mm filters into glass tubes.

8. Leave on ice for 4-5 min.

9. Reaction:

a. Aliquot 20 ml reaction mix/tube. Prepare a no-lysate tube for background control.

b. For MnSO₄ stimulation add 5 ml 200 mM stock to 20 ml reaction mix.

c. Add 200 ml lysate and shake gently.

d. Incubate 2 min at RT in a water bath.

e. Add 100 ml STOP solution.

f. Dilute to 1 ml with H₂O. Pipet 10 ml in counting vial for input calculation.

10. Chromatography:

a. Before assay wash columns

1. Dowex (yellow) twice with 10 ml of 1 mM imidazole pH 7.0 (blue label)

2. Alumina (white) twice with 10 ml of 100 mM imidazole pH 7.5 (orange label)

b. Transfer samples to Dowex column. Let soak in.

c. Wash twice with 1 ml of 1 mM imidazole pH 7.0.

d. Place Dowex columns on top of Alumina columns.

e. Wash into Alumina columns with 5 ml 1 mM imidazole pH 7.0.

f. Wash off Alumina columns into counting vials with 3 ml of 100 mM imidazole pH 7.5 and count 5 min.

g. Regenerate Dowex columns with 1 M HCl (use 2-3 ml).

Reaction Mix (=10X):

100 mM Tris pH 8.0

1 mM ATP

10 mM cAMP

100 mM DTT

0.5 ml a-³²P-ATP/tube

H20 to 20 ml

Lysis Buffer:

20 mM Tris pH 8

2 mM MgSO₄

for GTPγS stimulation add 80 mM GTPγS and 2 mM cAMP

This page was last edited 06/26/2003