Phosphoinositide 3-kinase phosphorylates the 3 position of Phosphatidylinositol (PI), Phosphatidylinositol-4 (PIP) and Phosphatidylinositol-4,5 (PIP2). PI-3 kinase is important in a number of signal transduction pathway such as survival and proliferation of endothelial cells (1,2). The activity of PI-3 kinase is measured by the amount of hot ATP incorporated into the lipid substrates, which are separated using thin layer chromatography (TLC).

1. Varticovski et al. Role of PI 3-kinase in mitogenesis. Biochinica et Biophysica Acta 1226 (1994): 1-11.
2. Fruman DA et al. Phosphoinositide kinases. Annu Rev Biochem 1998;67:481-507.

Lyuba Varticovski, M.D.(varticol@mail.nih.gov )
Staff Clinician
Office of the Director
National Cancer Institute
31 Center Drive, Room 3A11
Bethesda, MD 20892
T: (301) 496-4345
F: (302) 496-0775

5X PI Kinase buffer:
125mM MOPS pH 7.0
25mM MgCl2
5mM EGTA

Sonication buffer:
25mM MOPS pH 7.0
1mM EGTA

ATP Preparation:
cold ATP (-70°C stock, 0.2 or 0.1M)
For regular reaction, use 150-200 µM ATP using 2 mM ATP
For deacylation: dilute to 400 µM prior to use. Mix 0.001 µmoles cold ATP with 25 µCi 32P-ATP for each assay to be done.

Lipid:

PI:
L-a-Phosphatidylinositol
Bovine liver, sodium salt
Avanti Polar Lipids
Catalog Number 840042 in chloroform
aliquot as/is in brown glass vials, 1 ml, teflon tape on threads, seal top with Parafilm
(Note: do not use plastic tips to transfer chloroform solution)
2ml sample vials, Baxter B7791-2
Teflon-lined caps size 8-425, Baxter B7503-19

PI-4P:
L-a-Phosphatidylinositol 4-monophosphate
Bovine brain, sodium salt
Avanti Polar Lipids
Catalog Number 840045 in chloroform
dissolve in CHCl3:MeOH (2:1), at 1 mg/ml, aliquot as for PI above

PI-4,5P2:
L-a-Phosphatidylinositol 4,5-diphosphate
Bovine brain, sodium salt
Avanti Polar Lipids
Catalog Number 840046 in chloroform
dissolve in CHCl3:MeOH:1N HCl (2:1:0.01), at 1 mg/ml, aliquot as for PI above

PS
L-a-Phosphatidyl-L-Serine
Swine Brain
Avanti Polar Lipids
Catalog Number 840032P Powder
dissolve in CHCl3 at 10 mg/ml and aliquot as for PI above
Catalog Number 840032C in chloroform 10 mg/ml

Silica Gel 60 20x20 cm plates 250 m layer, such as Whatman from Fisher 05-713-165

TLC pretreatment solution:

1 mM EDTA and 1% (wt:vol) Potassium oxalate in methanol:water (40:60)

Oxalate pretreatment of TLC plates: Prepare fresh TLC pretreatment solution. Place plates in a TLC developing tank and add pretreatment solution by slow siphon. The speed should be such that the solvent front runs just ahead of the added solution. Be careful that the plates do not develop air pockets that do not get treated by the pretreatment solution. Air dry and then activate plates at100°C for 1 hr.

Before starting, label TLC plates and put in the oven to dry.

1. Prepare 300-500 ng protein per assay, however, if NP-40 or other detergent is present you must dilute it out at least 1/1000 times with 1X PI-3K buffer.
2. Prepare ATP (150 µM final in assay)
3. Prepare lipid (in regular Eppendorf tube

   a.	e.g. for 10 reactions, use 40µl PIP2, 4µl each of PI and PS (enough for 12 reactions) dry under nitrogen gas and add 120 µl sonication buffer. Seal the tube with parafilm to avoid evaporation.
   b.	Sonicate in water for 20 min for allowing miscelle formation.

4. Mix all reagents except ATP and lipids 5 min before sonication is done.

   a.	20µl	H2O
   b.	10µl	5X buffer
   c.	5µl	enzymes or whole cell lysate (diluted)
   d.	5µl	ATP
   e.	10µl	lipid
   f.	50µl	Total

5. Leave cap open and incubate in 37°C heat block. Tap tube every 5 min.
6. Prepare stop solution (1:1 MeOH:1N HCI) and stop the reaction with 90µl.
   
7. Extract the organic layer twice with 100µl CHCl3 and vortex gently. Use elongated tips to withdraw the lower layer to a new tube.
   REMEMBER: drop 1 drop back to the original tube and deposit all but one drop to the new tube. Combine the two organic layers and dry under nitrogen gas.
8. Add 15 ul of 2X CHCl3:1X MeOH solution to each tube, vortex and spin quickly.
9. Spot on TLC plate, no closer than 1 cm apart.
10. put TLC plate in TLC tank filled with running buffer (34ml water:65ml n-propanol:1ml glacial acid) and runovernight
11. expose with x-ray film. Cut out spots from TLC for counting radioactivity using scintillation counter.

• Make sure lipid extraction is as clean as possible; otherwise, a big smear of ATP will show up on TLC x-ray film.
• Do not put sonicated lipid on ice. Leave it at RT.
• When dealing with lipid, work as fast as possible to avoid evaporation