Guan Lab - Cornell Veterinary Medicine

Protocols - PI3K Assay Protocol

Appearance of phosphorylated lipid run on TLC.

Materials

Lipid substrate:

1. Dissolve phosphatidylinositol, and phosphatidyl serine (1:1) in buffer (25mM HEPES pH 7.4, 1 mM EDTA) to make a final concentration of 1 mg/ml.

2. Sonicate in a water bath sonicator for 5-10 mins.

3. Aliquot, and store at -80 °C for future use. After thawing out a tube, always vortex the lipid for 30 seconds before use.

1% Potassium oxalate (w/v) and 2 mM EDTA in H2O/MeOH (3:2) The 2 mM EDTA is the final concentration in water, not the final concentration in the solution. For example, 500 ml = 200 ml MeOH, and 300 ml 2 mM EDTA + 5 grams potassium oxalate.

Kinase Buffer: 10 mM MgCl2, 50 mM tris pH 7.4 (make fresh each time)

1 N HCl

CHCl3/MeOH (1:1)

MeOH/100 mM HCl, 2 mM EDTA (1:1) (the 100 mM HCl, 2 mM EDTA is one solution that is in a 1:1 ratio with MeOH)

CHCl3/MeOH/H2O/NH4OH (45:35:8.5:1.5) make this fresh each time, this sol'n may appear cloudy. It is important to be precise in mixing the components. A slight deviation in the ratios of each component will make the TLC run poorly.

silica gel 60 F254 plates, 0.2 mm thickness

Protocol:

The day before, pre-coat TLC plate in the oxalate solution at room temp. for 30 minutes in a large glass baking dish with gentle rocking. Air dry coated plate overnight. When you first add the coating solution, it may at first fizz a little and wet the plate unevenly. This will disappear by the time you're done coating. This step is not needed if the plate is pre-coated.

1. Incubate antibody w/ 0.5-1 mg lysate for at least 90 minutes (can be overnight). Add protein A sepharose beads and incubate for 1 hr.

2. Wash beads 3 times in lysis buffer, and once in 10 mM tris pH 7.4. Save an aliquot of the IP for Western blotting, use the rest for the kinase assay.

3. Incubate kinase assay part in 45 ul kinase buffer, 5 ul lipid substrate (1 mg/ml), and 10-20 uCi 32P-g-ATP. Mix gently, and keep at room temp. for 20 minutes.

4. Stop reaction by adding 100 ul of 1 N HCl.

5. Spin the samples for 1 min at 13000 rpm in microfuge.

6. Transfer supernatents to screw-capped tubes (this will keep your microfuge from becoming radioactive).

7. Add 160 ul of CHCl3/MeOH (1:1), briefly vortex (this step is for extracting the lipid).

8. Spin for 1 minute and discard the TOP layer (lipid is in the bottom layer).

9. Add 80 ul of MeOH/100 mM HCl, 2 mM EDTA (1:1) and vortex.

10. Spin for 1 minute, and discard the TOP layer (ATP will be in top layer).

11. Spot 5-10 ul on pre-coated TLC plate. Do not spot entire aliquot onto plate, as the liquid will spread out too much. Spot approx. 1 ul drops, waiting for each drop to dry (10-20 seconds) before adding more. You want to keep the spot within 5 mm in diameter. Your spots should be evenly spaced about 1 cm from the bottom of the plate. Allow the plate dry for 20 minutes. The remainder of each sample can be safely stored at -20°C.

12. Place TLC plate into TLC chamber and allow it to run for 20 - 30 minutes. You should use 90 ml of the TLC solvent.

13. Air dry the plate (5-10 minutes), wrap in plastic wrap and expose film overnight at -80 °C.

14. Develop the film and align it w/ the TLC plate. Cut out the migrated lipid spot and count the activity. Alternatively, subject the TLC plate to phoshoimager analysis.