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In Vitro Kinase Assay

Method

Note: One 10-cm dish per antibody (Ab) immunoprecipitation (IP) will give three kinase reactions per IP.

  1. Irradiate two 10-cm plates of HEK293 cells with 15 Gy and let recover for 30 minutes. Then lyse in 800 µl TGN buffer, scrape plates and transfer to an Eppendorf tube.
  2. Incubate on ice 20 minutes, then spin down cell debris, 10 minutes, 4°C, maximum speed. Pool all aliquots together to ensure equal concentration of protein.
  3. Aliquot 750 µl extract into one tube with no Ab. Aliquot another tube with 750 µl extract with 15 µl ATM/ATR Ab. Incubate 1 hour at 4°C, with rotation.
  4. At the same time, incubate two tubes with 45 µl Protein A beads/5% BSA in 45 µl TGN buffer each, for 1 hour at 4°C, to block.
  5. Quick-spin all tubes, low speed, quick pulse.
  6. After 1 hour, combine one tube lysate/Ab with one tube BSA/beads and incubate for another hour at 4°C.
  7. Resuspend each tube in 90 µl kinase buffer + 30 µCi β-32P-ATP. Then split into three separate tubes.
  8. Add 1 µg substrate (small volume).
  9. Incubate 30 minutes at 30°C.
  10. Add protein loading dye and run on gel (For GCN5, run 6-7% gel at 150 V.).
  11. Stain gel with Coomassie, destain (dispose of everything in radioactive waste), then dry on gel dryer and expose to film 1-4 hours. You can scan gel for a record of Coommassie.

Washes

  • Wash 2X TGN
  • Wash 1X RIPA
  • Wash 2X Kinase Buffer
  • Wash volume = 750 µl
  • Spin each at 5000g for 1-2 minutes

Solutions

TGN Buffer

  • 50mM Tris, pH 7.5
  • 50mM Glycerophosphate
  • 150mM NaCl
  • 10% Glycerol
  • 1% Tween-20
  • 1mM NaF
  • 1mM NaVO4
  • 1mM Phenylmethylsulfonylfluoride
  • 2 µg/ml Pepstatin A
  • 5 µg/ml Leupeptin
  • 10 µg/ml Aprotinin
  • 1mM DTT

Kinase Buffer

  • 10mM HEPES, pH 7.5
  • 50mM Glycerophosphate
  • 50mM NaCl
  • 10mM MgCl2
  • 10mM MnCl2
  • 5 µM ATP
  • 1mM DTT

© 2014 The University of Texas MD Anderson Cancer Center