Cellular kinases akt(thr308) april2011

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HTRF Phospho-Akt (Thr308) Data

Transcript of Cellular kinases akt(thr308) april2011

  • 1. HTRF cellular-kinase assays: for the simple detection of phosphorylated kinases directly in the cell
  • 2. HTRF Cellular kinase assays
    • OBJECTIVE:
      • Measurement of endogenous phosphorylated kinase in a cell-based format
    • TARGET:
      • Surface receptor investigation
      • Kinase Pathways
      • Screening of kinase modulators
    19/04/2011 Cellular Akt (Thr308)
  • 3. A simple, rapid and easy protocol: Activate the cells Detect the phosphorylated kinases 19/04/2011 Cellular Akt (Thr308)
  • 4. HTRF Cellular Kinase assays available Looking for other cellular Kinases? Let your scientific consultant develop this assay for you
    • Phospho-Erk(1/2) Cellulerk
    • Phospho Akt (Ser 473)
    • Phospho Akt (Thr 308)
    19/04/2011 Cellular Akt (Thr308)
  • 5. All assays designed with Simple Mix and Read Protocol Amenable to Miniaturization
    • Follow the viability of the cells using the Two-plate assay protocol Culture/Activation/Lysis in 96-well culture plate HTRF detection in white plate 384-well small volume
    • Use the One-plate assay protocol for HTS Culture/Activation/Lysis/HTRF detection in the same white plate 384-well small volume
    19/04/2011 Cellular Akt (Thr308)
  • 6. One-plate assay protocol Culture/Activation/Lysis/HTRF detection in the same white plate 384-well small volume Dispense 4L of ligand and incubate at room temperature Optimize the appropriate incubation time Plate 8L of cells in 384-well small volume-plate and incubate overnight, at 37C in CO2 atmosphere Dilute cells in growth medium using the appropriate cell density Add 2L of each HTRF conjugates and incubate for 2 hours at room temperature Add 4L of supplemented lysis buffer Incubate for 15min at room temperature on a plate shaker 19/04/2011 Cellular Akt (Thr308) 384-well SV plate
  • 7. Two-plate assay protocol Culture/Activation/Lysis in 96-well culture plate HTRF detection in white plate 384-well small volume Dispense 50L of ligand and incubate at room temperature Optimize the appropriate incubation time Plate 50L of cells in 96-well culture plate and incubate overnight, at 37C in CO2 atmosphere Dilute cells in growth medium using the appropriate cell density Remove carefully cell supernatant Add 50L of supplemented lysis buffer Incubate for 15min at room temperature on a plate shaker Transfer 16L of cell-lysate from cell-culture plate to a 384-well white plate small volume Add 2L of each HTRF conjugates and incubate for 2 hours at room temperature 19/04/2011 Cellular Akt (Thr308) 96-well culture plate plateplate 384-well SV plate 384-well SV plate 96-well culture plate plateplate
  • 8. The assays can be adapted to many plate configurations * Datas obtained with Phospho-Akt(Ser473) kit 19/04/2011 Cellular Akt (Thr308) 96-well plate SV 384-well plate regular 384-well plate regular 384-well plate SV Final volume 40l 40l 20l 20l Ref.PEH - Nb of tests/kit 5000 5000 10000 10000 Low control* 1170 1101 1411 968 High control* 4180 3970 4266 3719 window 3.6 3.6 3 3.8
  • 9. HTRF benefits
    • Homogeneous, mix & read:
    • no washing steps
    • time and labor saving
    • better robustness
    • Long signal stability:
    • no datas loss due to reader breakdown
    • time flexibility, read after an extended an extended period of time
    • assays can be measured as frequently as needed
    • Simple detection:
    • Readouts can be performed on > 20 possible instruments (BMG, Tecan, Molecular Devices, BioTek, Berthold Technologies)
    19/04/2011 Cellular Akt (Thr308)
  • 10. Phospho-Akt (Thr308) Assay in action
  • 11. Detection of Phospho-Akt on various cell-lines Cell lines Stimulators Inhibitors HEK Insulin, IGF A431 EGF CHO Insulin, IGF Wortmanine, PI-103
  • 12. Phospho-Akt(Thr308) EGF stimulation on A431 cell-line Two-plate assay protocol: Cells plated for 24 hours - Starving step: 24 hours after 2 PBS washes EGF stimulation (10) Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Cells/well 50,000 25,000 Top 3049 2874 bottom 1174 1068 window 2.6 2.7 EC50 1.72E-08 1.85E-08
  • 13. Phospho-Akt(Thr308) Insulin stimulation on HEK 293 cell-line Two-plate assay protocol: Cells plated for 24 hours - Starving step: 24 hours after 2 PBS washes insulin stimulation (10) Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Cells/well 50,000 25,000 Top 4238 4023 bottom 370 360 window 11.4 11.2 EC50 1.31E-08 1.72E-08
  • 14. Phospho-Akt(Thr308) IGF stimulation on HEK 293 cell-line Two-plate assay protocol: Cells plated for 24 hours - Starving step: 24 hours after 2 PBS washes IGF stimulation (10) Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Cells/well 25,000 Top 3981 bottom 363 window 11 EC50 1.1E-09
  • 15. Phospho-Akt(Thr308) IGF stimulation on CHO-M1 cell-line Two-plate assay protocol: Cells plated for 24 hours - Starving step: 24 hours after 2 PBS washes IGF stimulation (10) Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Cells/well 50,000 25,000 Top 6827 11367 bottom 1027 1862 window 6.6 6.1 EC50 0.98E-08 1.25E-08
  • 16. Phospho-Akt(Thr308) Insulin stimulation on CHO-M1 cell-line Two-plate assay protocol: Cells plated for 24 hours - Starving step: 24 hours after 2 PBS washes Insulin stimulation (10) Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Cells/well 50,000 25,000 Top 5530 9618 bottom 600 1402 window 9.2 6.8 EC50 1.49E-09 1.33E-09
  • 17. Phospho-Akt(Thr308) Inhibition of IGF stimulation on CHO cell-line Two-plate assay protocol: Cells (50,000) plated for 24 hours - Starving step: 24 hours after 2 PBS washes inhibition with wortmannine & PI-103 (5) - IGF stimulation (10) - Lysis (45) transfer of lysate addition of HTRF reagents - reading 19/04/2011 Cellular Akt (Thr308) Pathway inhibitor EC 50 PI-103 PI3K & mTor 4.44 10 -7 M Wortmannine PI3K 9.73 10 -7 M