PIKinase Assay

1. Add 50 iL TNE buffer to each immunoprecipitate and resuspend the beads by tapping the side of the microfuge tube with a finger. Quickly add the following: (1) 10 iL of the phosphoinositides that were resuspended in the TE buffer (2 mg/mL) as described above; (2) 10 iL of 100 mM MgCl2, and (3) 10 iL of the ATP solution (30 iCi 32P-ATP in 440 iM MgATP). Mix reagents by again tapping the side of the tube with a finger.

2. Allow the reaction to proceed for 10 min at room temperature (approx 22°C).

Fig. 2. Flowchart showing the major steps in the PI3K assay.

3. Stop the reaction by adding 20 iL of 8 M HCl followed immediately by 160 iL chloroform :methanol (1:1). The mixture will equilibrate into a lower organic phase and upper aqueous phase separated by a film containing the agarose beads.

4. Invert the tube to get good mixing; then, let it sit for several minutes at room temperature to allow the phases to separate. Ensure complete separation of the phases by centrifuging for 1 min at top speed in a microcentrifuge (approx 20,800g).

5. In a fume hood, transfer all of the lower phase to a new microfuge tube (see Note 9). This contains the 32P-labeled phosphoinositides, which can be loaded immediately on a TLC plate or stored overnight at -20°C.

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