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AC in vivo

 

 

IN VIVO ADENYLYL CYCLASE ASSAY

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

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.         Aliquot 2 ml of cells into small plastic cup and shake at ~200 rpm at RT.

8.         Zero time point:

- lyse 300 ml cells with 300 ml Lysis Buffer

- immediately assay 200 ml as in 11

9.         Add cAMP to 10 mM

10.       Do 60, 105, 180, and 300 minutes time points as in 8.

11.            Reaction:

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

  control. Do assay in duplicate.

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

- Add 200 ml lysate and shake gently.

- Incubate 1 min at RT in a water bath.

- Add 100 ml STOP solution.

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

12.            Chromatography:

- Before assay wash columns

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

- Alumina (white) twice with 10 ml of 100 mM imidazole pH 7.5 (orange

   label)

- Transfer samples to Dowex column. Let soak in.

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

- Place Dowex columns on top of Alumina columns.

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

- Wash off Alumina columns into counting vials with 3 ml of 100 mM imidazole

   pH 7.5 and count 5 min.

- 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

 
Last modified:02/01/00