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PI (phosphoinositide)-3 Kinase Assay

HP Huan Pang

Published: Apr 20, 2012 DOI: 10.21769/BioProtoc.203 Views: 10747

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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

Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.

Category

Biochemistry > Protein > Activity

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Why do we need to add extra ATP in addition to...
edit 0 Answer 9 Views Sep 15, 2014