cellular kinases akt(thr308) april2011

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

219/04/2011 Cellular Akt (Thr308)

A simple, rapid and easy protocol:Activate the cells

Detect the phosphorylated kinases


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)


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


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


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


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


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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)


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

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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


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

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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


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

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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


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


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

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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


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


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


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


cellular kinases akt(thr308) april2011

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