1. Metabolically label cells with 32P (see Protocol on Metabolic Labeling of Proteins with 32Phosphate).
2. Collect labeled and washed cell pellet in a 15 ml conical tube.
3. Add 240 μl of ddH2O and 60 μl of 10% SDS.
4. Incubate suspension in a boiling water bath for 5 min (see Hint #2).
5. Transfer the supernatant to a microcentrifuge tube.
6. Add 100 μl of 2% SDS to the cell pellet.
7. Boil for 3 min and pool this cell pellet supernatant with the supernatant from Step #5.
8. Centrifuge in a microcentrifuge at room temperature for 2 min at maximum speed to clarify the solution.
9. Transfer the supernatant to a microcentrifuge tube with a screw cap.
10. Add 300 μl of Saturated Phenol and mix well by vortexing for 30 sec.
11. Centrifuge in a microcentrifuge for 2 min at maximum speed and remove the aqueous phase (see Hint #3).
12. To the aqueous phase, add an equal volume of Saturated Phenol, mix well by vortexing for 30 sec, centrifuge in a microcentrifuge at maximum speed for 2 min, and discard the aqueous phase.
13. Pool the phenol and interface solutions.
14. To the pooled phenol solution, add 450 μl of Tris Buffer, mix well by vortexing, and centrifuge in a microcentrifuge for 2 min at maximum speed.
15. Repeat the Tris Buffer washing of the phenol phase 2 more times (Step #14).
16. Add the phenol and interface to a 30 ml Corex tube.
17. Add 20.4 ml of ddH2O (see Hint #4).
18. Add 20 μl of γ-Globulin and 3.6 ml of 100% Trichloroacetic acid.
19. Cover tube opening with Parafilm and vortex briefly.
20. Incubate solution overnight (18 hr) in an ice-bath.
21. Centrifuge at 0°C for 40 min at 16,500 X g (10,000 rpm using an HB-4 rotor).
22. Carefully remove the supernatant with a Pasteur pipette.
23. Wash the pellet with Chloroform:Methanol Solution at room temperature (mix well).
24. Centrifuge at 16,500 X g for 25 min at 0°C (10,000 rpm using an HB-4 rotor).
25. Discard the supernatant.
26. Wash the pellet with 5 ml of 100% Ethanol at room temperature (mix well).
27. Centrifuge at 16,500 X g for 25 min at -10°C (10,000 rpm using an HB-4 rotor).
30. Discard the supernatant.
31. Wash the pellet with 5 ml of 75% Acetone at room temperature (mix well).
32. Centrifuge at 16,500 X g for 25 min at -10°C (10,000 rpm using an HB-4 rotor).
33. Discard the supernatant.
34. To the pellet add 500 μl of 6 N HCl.
35. Incubate in boiling water bath for 1 min.
36. Transfer the solution to microcentrifuge tube and place in an ice bath.
37. Rinse the Corex tube 3X using 100 μl of 6 N HCl and pool each wash solution into the microcentrifuge tube placed on ice.
38. Hydrolyze at 110°C for 90 min in microcentrifuge tube (with cap locked) (see Hint #5).
9. Place the microcentrifuge tube on ice.
40. Add 500 μl of ddH2O.
41. Evaporate the solution using a vacuum microcentrifuge concentrator.
42. Resuspend the residue in 13 μl of pH 1.9 Buffer.
43. Remove 1 μl, add to 10 ml scintillation fluid, and count in a scintillation counter.
44. Load sample on a 0.1 mm cellulose TLC plate (EM Sciences) by applying approximately 0.5 μl (see Hint #6). Include 1 μg of each of the PAA Standards.
45. Carry out two-dimensional (2D) chromatography (see Protocol on Two-Dimensional (2D) Thin Layer Chromatography).
46. Expose dried TLC plate to film for autoradiography. Assume that 5 X 106 cpm is equivalent to 1 day exposure with an enhancing screen.
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