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PI (phosphoinositide)-3 Kinase Assay
PI肌醇磷脂3-激酶活性测定   

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Abstract

PI3-kinases regulate a wide range of cellular responses through the production of phosphatidylinositol 3, 4, 5-trisphosphate [PI (3, 4, 5) P (3)] in cellular membranes. This protocol provides a highly efficient and easy-to-handle method for sensitive detection and quantification of PI 3-kinase activity.

Materials and Reagents

  1. Tris
  2. NaCl
  3. EDTA
  4. Triton-X100
  5. Glycerol
  6. NaF
  7. PMSF
  8. Aprotinin
  9. Leupeptin
  10. Sepharose
  11. NP-40
  12. Tris-LiCl
  13. TNE
  14. P32 hot ATP (PerkinElmer, catalog number: NEG502A )
  15. MgCl2
  16. MnCl2
  17. EGTA
  18. Methanol
  19. Chloroform
  20. Acetic acid
  21. Isopropanol
  22. Lysis buffer
  23. Pre-washed sepharose
  24. ATP mix (see Recipes)
  25. Phosphoinositol lipid (Avanti Polar Lipid, catalog number: 190082 ) (see Recipes)

Equipment

  1. Centrifuges
  2. Sonicator
  3. Radiation hood
  4. Silica gel TLC plate

Procedure

  1. Lyse the cell with cantley lysis buffer plus protease inhibitor (aprotinin 100 μg/ml, Leupeptin 1 μg/ml, PMSF 35 mg/ml), spin at 10,000 x g for 15 min.
  2. Take the supernatant after centrifugation. Immunoprecipitate with the antibody against protein of interest at 4 °C on wheel overnight.
  3. Add 60 μl pre-washed sepharose (either protein A or protein G) with cut pipet tips to each sample. Put on wheel at 4 °C for 2-3 h.
  4. Spin the sample, (you might want to save the supernatant for future analysis). Wash the samples extensively with following:
    1. 3x 1 ml wash with PBS-1% NP-40
    2. 3x 1 ml wash with Tris- LiCl
    3. 2x 1 ml wash with TNE
    Make sure the washes will not sit at room temperature (RT) for long. Add 60 μl TNE to each sample.
  5. Return the samples on ice while preparing substrates for the assay:
    For each sample phosphoinositol lipid, ATP mix and 100 mM MgCl2 or MnCl2 is needed.
  6. After preparing all the reagents, add 10 μl of PI lipid and 10 μl of 10 mM MgCl2 to each sample. Now you are ready to perform the assay in the radiation hood.
  7. Add hot ATP in the hood. Start the assay by adding 5 μl ATP mix to the sample 10 sec apart and stop each reaction with 20 μl of 8 M HCl when reach 10 min.
    Note: make sure to mix the reaction well for each addition.
  8. Add 160 μl of 1:1 methanol: chloroform to extract lipid and mix well.
  9. Spin the samples in the hot centrifuge for 5 min, remove the upper (water) phase.
  10. Wash the organic layer twice with 200 μl 1 M HCl.
  11. Spot adequate amount of washed chloroform phase onto a silica gel TLC plate (the sample can be stored at -70 °C if not spotting).
  12. Run the plate in the solvent mix (2 M acetic acid/isopropanol 1:2) tank until the solvent front is about 2-3 cm away from the top.
  13. Take out the plate and dry in the hood.
  14. Wrap the plate and expose on the film at -70 °C overnight.
  15. Quantify the phosphatidylinositol3-phosphate spots.

Recipes

For example, if you have 10 samples prepare the reagents as following: Put two extra in: 10+2= 12 samples.

  1. PI lipid
    2 μl x 12 samples= 24 μl
    Take out 24 μl PI, dry under the Argon. Bring up to 120 μl.
    10 mM Tris and 1 mM EGTA.
    Sonicate 10 min.
  2. ATP Mix
    Need total volume 5 μl x 12 samples= 60 μl ATP Mix
    12 μl hot ATP (10 uCi/μl)
    12 μl Cold 10 mM ATP
    36 μl H2O
  3. MgCl2
    10 μl x 12 samples= 120 μl total

References

  1. Wu, H., Shekar, S. C., Flinn, R. J., El-Sibai, M., Jaiswal, B. S., Sen, K. I., Janakiraman, V., Seshagiri, S., Gerfen, G. J., Girvin, M. E. and Backer, J. M. (2009). Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants. Proc Natl Acad Sci U S A 106(48): 20258-20263.
  2. Yip, S. C., El-Sibai, M., Hill, K. M., Wu, H., Fu, Z., Condeelis, J. S. and Backer, J. M. (2004). Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells. Cell Motil Cytoskeleton 59(3): 180-188.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Pang, H. (2012). PI (phosphoinositide)-3 Kinase Assay. Bio-101: e203. DOI: 10.21769/BioProtoc.203.
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salil sukumaran
Institute for Biochemistry I
Why do we need to add extra ATP in addition to Gamma-P32-ATP?
2014/9/15 4:12:14 回复