Cell-based kinase assays in HTS Benjamin Bader

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HTRF – assay HTRF study goals: • set up cell-based mTOR kinase activity assay for HTS using pAkt Ser473 readout • identify optimal cancer cell background • validate with reference inhibitors Detection (modified from Carlson 2009) (from Cisbio product insert) 20 HTRF-symposium 2013 201 20
HTRF – cell lines 18000 Ratio 16000 14000 12000 10000 8000 6000 4000 continuous culture frozen cells • MCF7 cells strongest S/B • frozen cell assay • suspension cell format • miniaturized to 1536 well 2000 0 10 -10 10 -9 10 -8 10 -7 10 -6 AZD 8055 [M] 21 HTRF-symposium 2013 211 21
Page 1 and 2: Cell-based kinase assays in HTS Pot
Page 3 and 4: Kinases as drug targets cAbl and Gl
Page 5 and 6: Features of a typical early kinase
Page 7 and 8: General options for cell-based kina
Page 9 and 10: TR-FRET systems: HTRF and Lanthascr
Page 11 and 12: Introducing the PI3K / Akt / mTOR p
Page 13 and 14: Lanthascreen Assay Development BacM
Page 15 and 16: Lanthascreen - virus efficiency 30%
Page 17 and 18: Lanthascreen - optimization • sti
Page 19: Lanthascreen - inhibitor validation
Page 23 and 24: HTRF - pathway inhibitor validation
Page 25 and 26: Focussed medium throughput screen 2
Page 27 and 28: Focussed medium throughput screen L
Page 29 and 30: Acknowledgements Anja Kretzschmar S

Cell-based kinase assays in HTS Benjamin Bader

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