Molecular Techniques and Methods

In vivo Protein Phosphorylation in Mammalian Cells

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Tris/Saline Solution (1 liter)
0.15 M CaCl2 ----------------------------------------------------- 150 ml of 1 M CaCl2
20 mM Tris-HCl (pH 7.5) ----------------------------------------- 20 ml of 1 M Tris-HCl
Deionized H2O ---------------------------------------------------- 830 ml


Lysis Buffer (100 ml)
10 mM Tris-HCl (pH 7.5) ---------------------------------------- 1 ml of 1 M Tris-HCl
2 mM EDTA ----------------------------------------------------- 0.4 ml of 0.5 M EDTA
2 mM PMSF ----------------------------------------------------- 20 ml of 10 mM PMSF
3% SDS ---------------------------------------------------------- 30 ml of 10% SDS
0.1% NP-40 ------------------------------------------------------ 0.2 ml 50% NP-40
10 mM NaCl ------------------------------------------------------ 0.2 ml of 5 M NaCl
0.15 mM MgCl2 -------------------------------------------------- 15 ul of 1 M MgCl2
Deionized H2O ---------------------------------------------------- 48 ml




PROCEDURES

1. Grow mammalian cells in DME medium overnight.

2. Wash cells twice with DME medium without phosphate.

3. Incubate cells with DME medium containing 200 uCi/ml of 32Pi for 3-6 hours at 37oC to label endogenous ATP pool and protein.

4. Teminate the reaction by washing the cells with ice-cold Tris/Saline Solution three times.

5. Incubate cells with 5% TCA for 20 minutes.

6. Rinse cells with ice-cold Tris/Saline Solution two times.

7. Add 200-400 ul of Lysis Buffer immediately.

8. Centrifuge cellular lysates at 14,000g and 4oC for 10 minutes.

9. Collect the supernatant.

10. Measure protein concentration by BCA method.

11. Boil 50 ug protein in 10-20 ul for 10 minutes.

12. Analyze protein phosphorylation in 5-12% SDS-PAGE.


NOTES



KIT INFORMATION



REFERENCES

  • Huang, Y.-T., Kuo, M.-L., Liu, J.-Y., Huang, S.-Y., and Lin, J.-K., 1996, Inhibitions of Protein Kinase C and Proto-oncogene Expressions in NIH 3T3 Cells by Apigenin. J. of Cancer Research.

  • Rodriguez-Pena, A., and Rozengurt, E., 1986, Phosphorylation of an acidic mol. wt. 80,000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity. The EMBO J. 5, 1, 77-83.



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