1. grow cells to 5 x 10⁶ cells/ml

2. develop for 5 hours at 2 x 10⁷ cells/ml in DB with 50 nM cAMP pulses

3. basalate in 2 mM caffeine/DB for 30 min

4. wash in PM and resuspend at 8 x 10⁷ cells/ml in PM; leave cells on ice

5. Zero time point:

- lyse 150 ml cells with 150 ml lysis buffer into 1.5 ml ice cold PM Buffer

- spin for 1 min at RT (max speed in microfuge)

- aspirate supernatant (well)

- resuspend with 60 ml 1X Sample Buffer

- load 10-20 ml on gel

- perform Western Analysis

6. Aliquot cells into small plastic cup and shake at 200 rpm

7. Add cAMP to 10 mM

8. Do 5, 20, 60, and 180 secs time points as in 5.

This page was last edited 06/26/2003