In Vivo Actin Polymerization ( cAMP)

 

Chemicals and equipments

Actin Buffer ( 20 mM KPO4, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8), Assay buffer (Actin Buffer with 3.7 formaldehyde, 0.1 triton, 200X TRITC- Phaloidin from 50 mM stock.), DB,

Prepare Cells

a. Wash cells with DB and resuspend them to 2 X 107 / ml inDB

b. Shake cells at 120 rpm for 1 hour

c. Pulse cells with 100 nM cAMP for 4 hours

d. Basolate cells with 3 mM caffeine for 15-30 min

e. Spin cells down, add DB + 3 mM caffeine to 3 X 107 /ml, shake 200 rpm for 10 min.

Assay

Take 2 ml cells, shake at 200 rpm, at time 0 shoot 0.2 ml 1 mM cAMP

At time 0, 4", 8" 15", 30", take 100 ml aliquot out to 1ml assay buffer.

Cover with aluminum foil, shake to fix at room temperature for 1 hour.

Spin 14 K for 10 min, keep the pellet.

Add 1 ml MeOH, wrap in Aluminum foil, shake overnight.

Read sample on Fluro-Max

(excitation 3 mm 525, emission 5 mm 564)