cellular kinases akt(thr308) april2011
DESCRIPTION
HTRF Phospho-Akt (Thr308) DataTRANSCRIPT
HTRF® cellular-kinase assays:for the simple detection of phosphorylated kinases
directly in the cell
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
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A simple, rapid and easy protocol:Activate the cells
Detect the phosphorylated kinases
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HTRF® Cellular Kinase assays available
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Looking for other cellular Kinases? Let your scientific consultant develop this assay for you
Phospho-Erk(1/2) – Cellul’erk Phospho Akt (Ser 473) Phospho Akt (Thr 308)
19/04/2011 Cellular Akt (Thr308)
Follow the viability of the cells using the Two-plate assay protocolCulture/Activation/Lysis in 96-well culture plate HTRF® detection in white plate 384-well small volume
Use the One-plate assay protocol for HTSCulture/Activation/Lysis/HTRF® detectionin the same white plate 384-well small volume
All assays designed with Simple Mix and Read Protocol
Amenable to Miniaturization
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Dispense 4µL of ligand and incubate at room temperatureOptimize the appropriate incubation time
Plate 8µL of cells in 384-well small volume-plate and incubate overnight, at 37°C in CO2 atmosphereDilute cells in growth medium using the appropriate cell density
Add 2µL of each HTRF conjugates andincubate for 2 hours at room temperature
Add 4µL of supplemented lysis bufferIncubate for 15min at room temperature on a plate shaker384-well SV plate
One-plate assay protocolCulture/Activation/Lysis/HTRF® detection
in the same white plate 384-well small volume
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Dispense 50µL of ligand and incubate at room temperatureOptimize the appropriate incubation time
Plate 50µL of cells in 96-well culture plate and incubate overnight, at 37°C in CO2 atmosphereDilute cells in growth medium using the appropriate cell density
96-well culture plate plateplate
384-well SV plate
384-well SV plate
96-well culture plate plateplate
Remove carefully cell supernatant
Add 50µL of supplemented lysis bufferIncubate for 15min at room temperature on a plate shaker
Transfer 16µL of cell-lysate from cell-culture plate to a 384-well white plate small volume
Add 2µL of each HTRF conjugates andincubate for 2 hours at room temperature
Two-plate assay protocolCulture/Activation/Lysis in 96-well culture plate
HTRF® detection in white plate 384-well small volume
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The assays can be adapted to many plate configurations
96-well plate SV
384-well plate regular
384-well plate regular
384-well plate SV
Final volume 40µl 40µl 20µl 20µl
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
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* Datas obtained with Phospho-Akt(Ser473) kit
19/04/2011 Cellular Akt (Thr308)
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HTRF® benefits
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Homogeneous, mix & read: no washing stepstime and labor savingbetter robustness
Long signal stability:
no datas loss due to reader breakdowntime flexibility, read after an extended an extended period of timeassays can be measured as frequently as needed
Simple detection: Readouts can be performed on > 20 possible instruments (BMG,
Tecan, Molecular Devices, BioTek, Berthold Technologies)
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Phospho-Akt (Thr308)
Assay in action
Detection of Phospho-Akt on various cell-lines
Cell lines Stimulators Inhibitors
HEK Insulin, IGF
A431 EGF
CHO Insulin, IGF Wortmanine, PI-103
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Phospho-Akt(Thr308)EGF stimulation on A431 cell-line
Stimulation with EGFon A431 cells
-15 -120
500
1000
1500
2000
2500
3000
3500
50,000 cells
25,000 cells
-11 -10 -9 -8 -7 -6 -5
log [EGF] (M)
Rat
io H
TR
F
Cells/well 50,000 25,000
Top 3049 2874
bottom 1174 1068
window 2.6 2.7
EC50 1.72E-08 1.85E-08
12
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
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Phospho-Akt(Thr308)Insulin stimulation on HEK 293 cell-line
Stimulation with insulinon HEK 293 cells
incubation 4h
-12 -11 -10 -9 -8 -7 -6 -50
1000
2000
3000
4000
50,000 cells25,000 cells
log [insulin] (M)
Rat
io H
TR
F
Cells/well 50,000 25,000
Top 4238 4023
bottom 370 360
window 11.4 11.2
EC50 1.31E-08 1.72E-08
13
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
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Phospho-Akt(Thr308)IGF stimulation on HEK 293 cell-line
Stimulation with IGFon HEK 473
-13 -12 -11 -10 -9 -8 -7 -6 -50
500
1000
1500
2000
2500
3000
3500
4000
4500
25,000 cells
log[IGF] (M)
Rat
io H
TR
F
Cells/well 25,000
Top 3981
bottom 363
window 11
EC50 1.1E-09
14
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
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Phospho-Akt(Thr308)IGF stimulation on CHO-M1 cell-line
Stimulation with IGFon CHO-MR1 cells
incubation 4h
-12 -11 -10 -9 -8 -7 -6 -50
2000
4000
6000
8000
10000
12000
50,000 cells25,000 cells
log [IGF] (M)
Rat
io H
TR
F
Cells/well 50,000 25,000
Top 6827 11367
bottom 1027 1862
window 6.6 6.1
EC50 0.98E-08 1.25E-08
15
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)
Phospho-Akt(Thr308)Insulin stimulation on CHO-M1 cell-line
Stimulation with insulinon CHO-MR1 cells
-12 -11 -10 -9 -8 -7 -6 -50
3000
6000
9000
12000
50,000 cells25,000 cells
log [insulin] (M)
Rat
io H
TR
F
Cells/well 50,000 25,000
Top 5530 9618
bottom 600 1402
window 9.2 6.8
EC50 1.49E-09 1.33E-09
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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
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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
Phospho-Akt(Thr308) Inhibition of IGF stimulation on CHO cell-line
Phospho-Akt (Thr308)Inhibition of IGF stimulation
on CHO-CHMR1
-11 -10 -9 -8 -7 -6 -5 -4 -30
3000
6000
9000
Wotmannine
Pi 103
log[inhibitor] (M)
Ra
tio
HT
RF
Pathway inhibitor
EC50
PI-103 PI3K & mTor 4.44 10-7 M
Wortmannine PI3K 9.73 10-7 M
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