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Isolation of 32P-labeled Protein for Phosphoamino Acid and Phosphopeptide Analyses.

Isolation of 32P-labeled Protein for Phosphoamino Acid and Phosphopeptide Analyses.
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
This protocol explains the procedure for isolation of radiolabeled protein from polyacrylamide gels in preparation for phosphoamino acid or phosphopeptide analyses. Gel purification of the samples is necessary to avoid contamination with other polypeptides that could confound the analysis of the phosphoamino acid and/or phosphopeptide profiles of the phosphoprotein of interest.
1. Prepare samples of 32P-labeled protein either by metabolically labeling cultured cells with 32P-orthophosphate or by labeling recombinant or purified protein samples with protein kinase in vitro. For metabolically labeled cells, immunoprecipitate the protein of interest and resuspend the precipitate in SDS-PAGE (Laemmli) sample buffer and store at -80 °C until ready to use.

2. Subject the sample to SDS-PAGE to resolve individual polypeptides (see Protocol on Casting, Preparing and Running SDS-Polyacrylamide Gels).

3. Following electrophoresis, remove the gel from the electrophoresis apparatus and place onto a piece of 3 M Whatman paper. Cover the gel with plastic wrap and dry the gel on a gel drier with high heat (80°C) for one to two hours.

4. Expose the dried gel to X-ray film at -80°C in a cassette with an intensifying screen for several hours to visualize the radiolabeled protein(s) in the gel. Use radiolabeled marker dots or luminescent markers in order to align the gel with the autoradiogram following exposure and development (see Hint #2).

5. Align the film and the dried gel and staple the two together. Pierce the film and cut an area of the gel corresponding to the protein band on the autoradiogram. While holding the gel piece with a pair of tweezers, remove as much of the Whatman paper backing as possible without disrupting the integrity of the dried gel slice. The paper fibers can interfere with the subsequent analyses (see Hint #3).

6. Measure the Cerenkov counts in a scintillation counter. Do NOT use scintillation fluid!

7. Prepare fresh 50 mM NH4HCO3 and adjust the pH of the solution to between 7.3 and 7.6.

8. Add 500 μl of the 50 mM NH4HCO3 solution to the dried gel slice in a 1.5 ml microcentrifuge tube and incubate for 5 min at room temperature to rehydrate the gel.

9. Grind the gel piece with a spatula or pipette tip until it is able to pass through a P200 pipette tip.

10. Transfer the gel suspension to a 1.5 ml screwcap microcentrifuge tube. Rinse the original tube twice with 250 μl of 50 mM NH4HCO3 and transfer the solutions to the 1.5 ml tube to ensure that the sample is fully recovered.

11. Add 50 μl of 2-mercaptoethanol and 10 μl of 10% SDS, and boil the sample for 5 min.

12. Incubate with shaking overnight at 37°C to elute the protein from the gel.

13. Vortex the sample and centrifuge for 2 min at 2,500 rpm in a microcentrifuge.

14. Transfer the supernatant to a fresh 1.5 ml screwcap microcentrifuge tube.

15. Add 50 mM NH4HCO3 to the pellet to a final volume of 1.2 ml.

16. Add 50 μl of 2-Mercaptoethanol and 10 μl of 10% SDS, boil the sample for 5 min and incubate for 1 hr at 37°C.

17. Prepare the Performic Acid Solution and 100% TCA and place both on ice until ready to use.

18. Vortex the sample and spin for 2 min at 2,500 rpm in a microcentrifuge. Collect the supernatant into the same tube as Step #14.

19. Add 20μg of BSA and 250μl of ice cold 100% TCA to the tube and incubate for 1 hr to precipitate the sample.

20. Centrifuge at maximum speed in a microcentrifuge for 10 min at 4°C and remove the supernatant, leaving approximately 100 μl behind.

21. Repeat Steps #19 and #20 and remove all residual TCA from the pellet.

22. Add 500 μl of 100% Acetone chilled to -20°C to the pellet and microcentrifuge at top speed for 5 min at 4°C.

23. Remove the supernatant and air dry the pellet. Measure the Cerenkov counts again in the scintillation counter to determine the recovery of the radiolabeled protein.

24. Resuspend the pellet in 50μl of Performic Acid Solution and incubate on ice for 60 min (see Hint #4).

25. Immediately following Step #24, add 400 μl of ddH2O and vortex.

26. Transfer a fraction of the sample corresponding to approximately 1,000 cpm to a fresh tube for the phosphoamino acid analysis, leaving the majority of the sample for the phosphopeptide analysis.

27. Freeze the sample in a dry ice/ethanol bath, and lyophilize until dry (about three to four hr).

28. Store the samples at -80°C until ready to use.

10% (w/v) Sodium Dodecyl Sulphate (SDS) in ddH2O   in ddH2O
14.1 M 2-Mercaptoethanol
Performic Acid Solution (CAUTION! see Hint #1)   100 μl of 30% (v/v) Hydrogen Peroxide
900 μl of 98% (v/v) Performic Acid
100% Trichloroacetic Acid Solution (TCA) (CAUTION! see Hint #1)   total volume will be 5 ml
prepared in 2.27 ml ddH2O
5 g Trichloroacetic Acid
50 mM Ammonium Bicarbonate (50 mM NH4HCO3) Solution
50 mM Ammonium Bicarbonate (50 mM NH4HCO3) Solution   400 mg NH4HCO3
prepared in 100 ml ddH2O
BioReagents and Chemicals
Trichloroacetic Acid
Hydrogen Peroxide
Ammonium Bicarbonate
[32P] Orthophosphate
Performic Acid
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.

2. The length of exposure depends upon specific activity of label and the amount of sample applied to the gel and must be determined empirically.

3. The sample may be frozen at this stage.

4. This step fully oxidizes the methionine and cysteine residues so that they are in only one state during chromatography.