INVESTIGATING KINASE ACTIVITY IN A CELLULAR
CONTEXTE.TrinquetVP Product DevelopmentSLAS meeting, Cisbio Tutorial January 2013, Orlando
Protein phosphorylations are key signalling events
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Phospho-proteins
Signalling cascades
Cellular responses(proliferation, cell death…)
Dysregulationmutation
Diseases(cancers, inflammation…)
Common techniques to monitor protein phosphorylation
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Western Blot
ELISA ELISA-like techniques
Homogeneousbead-based techniques
Cell lysates
A straightforward HTRF® assay platform to monitor proteinphosphorylation using adherent or suspended cells
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HTRF assays are fast & convenient compare to othertechniques
Mix & Read
Time & Labor Saving
BetterRobustness
HTRF® & WB are well correlated, but HTRF® oftendisplays a superior sensitivity
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39 19 9.6 4.8 2.4 1.2 0.6 0.3 0.150
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X 103 cells
HTRF assay
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hosp
ho-M
EK
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MEK1 (45kDa)
Western blot (ECL reagents)
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r² 0.9715
Phospho-MEK1
HTRF Phospho-MEK1 signal (Delta F%)
Wes
tern
blo
t in
tens
ity (
EC
L)
Phospho-MEK1 (Hela cells)Sensitivity comparison
(cells / well)
Cellular kinase HTRF Western-blot
P-ERK 200 1960
P-AKT 195 3125
P-Stat3 750 750
P-p38 MAPK 1250 10000
P-IKKb 580 46500
P-mTOR 1250 1250
P-CREB 6250 6250
P-MEK1 600 2400
P-MEK1/2 1200 1200
P-JNK 3120 3120
P-c Jun 2500 20000
HTRF® & ELISA show similar results & sensitivity
Experimental conditions Assay Window Compound EC50 (M)
Cellular kinase assay Cell line Compound HTRF ELISA HTRF E LISA
P-Stat3 (Y705) Hela IFNa 7.7 5.3 10.7E-10 5.9E-10
P-p38 MAPK (T180-Y182) Hela anisomycin 10.8 3.5 7.6E-8 8.7E-8
P-IKKb (Ser177/181) Hela IL1b 3.5 3.6 2.48E-11 3.58E-11
P-mTOR (S2448) HEK293 rapamycin 2.4 2.2 5.5E-6 4.7E-6
P-CREB (S133) HEK293 Forskolin 4.1 4.6 1.4E-6 1.1E-6
P-MEK1 (S217/S222) Hela EGF 3.4 2.8 7E-10 1E-9
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log [IL1 ββββ] M
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.D. 450nm
Phospho-IKKb (Hela cells)
HTRF® is the most sensitive homogeneous technique to monitor protein phosphorylation
Experimental conditions Assay Window Compound EC50 (M)
Cellular kinase assay Cell line Compound HTRF Surefire® H TRF Surefire®
P-Stat3 (Y705) Hela IFNa 7.7 3 10.7E-10 1.3E-9
P-p38 MAPK (T180-Y182) Hela anisomycin 10.8 13.5 7.6E-8 6.0E-8
P-IKKb (Ser177/181) Hela IL1b 3.5 1.4 2.48E-11 1.79E-11
P-mTOR (S2448) HEK293 rapamycin 2.4 1 5.5E-6 -
P-CREB (S133) HEK293 Forskolin 4.1 2.5 1.4E-6 3.6E-9
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log [anisomycin] (M)
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Surefire signal
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log [IL1 ββββ] M
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Surefire signal
Phospho-IKKb (Hela cells)Phospho-p38 (Hela cells)
High reagents quality enables long-term programs
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Batch to batch reproducibility(Phospho-P38 – HeLa cells)
Reagent stability
Freeze/Thaw tolerance(Phospho-IKKb – HeLa cells)
Long term stability(Phospho-mTor – HEK293)
No Lys
ate 1/8
1/4
1/2
Pure l
ysate
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ate 1/8
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sate
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+3 Months
QC after production
+ 6 Months
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lysa
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Fresh1 Freeze/Thaw cycle2 Freeze/Thaw cycles
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Cell background compatibility
• HTRF® assays are usually compatible with human, mouse & rats cell lines
• HTRF® assays can work either with adherent or suspended cells
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log [Erlotinib] M
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log [FSK] M
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log [FSK] M
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P-CREB Human cells (A431)
P-CREB Mouse cells (NIH3T3)
P-CREB Rat cells (PC12)
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log [Anisomycin] (M)
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log [IL1-beta] (M)
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P-JNK Adherent cells (HEK293)
P-JNK Suspended cells (Jurkat)
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Flexibility in assays format
Photo courtesy of Greiner Bio-One
384 sv well20µl
96 well50µl
Phosphorylation StepVolume of cell lysates
Detection StepVolume of detection
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Flexibility in assays format
Photo courtesy of Greiner Bio-One
96 Half well40µl
384 sv well20µl
1536 well5-10µl
96 well50µl
6 well500µl
24 well250µl
T1753-5ml
T751.5ml
T250.5ml
Ø10cm1.5ml
Ø3.5cm500µl
384 well20µl
Phosphorylation StepVolume of cell lysates
Detection StepVolume of detection
Assay robustness
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Assay equilibrium
Assay equilibrium + 20H
[IFN alpha]M
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Assay equilibrium
Assay equilibrium + 4H
Assay equilibrium + 20H
[IFN alpha]M
Sur
efire
sig
nal
P-STAT3 assay on HeLa Cells
HTRF Surefire®
• HTRF® signal shows no degradation over time
• HTRF® allows multiple readouts
HTRF® assays are amenable to HTS
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log [PMA] (M)
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Phospho-MEK1Jurkat Cells – 20kcells/well
• Flexible use of HTRF kits due to high reagent quality
• Cell background compatibility
• Flexibility in assays format
• Assay robustness
• HTS amenable
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HTRF® Phospho assay main feature allow their use under a large variety of formats for drug research
Total protein detection is now possible with the sameplatform
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• Accurate determination of compound MOA- Assessment of protein expression- Assessment of protein phosphorylation
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log [EGF] M
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spho
ryla
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Total P
roteinH
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F R
atio
Phospho & Total ERKA431 cells – 5’ EGF
Phospho & Total protein detection can be done with the same cell lysate
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HTRF® is as sensitive as WB to monitor total protein level
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StimulatedUnstimulated
Blk 586 1172 2344 4688 9375 18750 37500 75000 1500000
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StimulatedUnstimulated
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Blk 586 1172 2344 4688 9375 18750 37500 75000 1500000
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StimulatedUnstimulated
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Phospho-ERK 1/2 - A431
Cells/well
Total ERK 1/2 - A431
Sensitivity comparison(Number of cells)
Target HTRF WB
Stat3 1172 1172
MEK1 9375 4688
ERK 1/2 1172 4688
Total proteins can be quantitatively detected in severalspecies
• Validated on human, mouse, rat and hamster cell lines
• Quantitative protein detection
Negative A431 Hela NIH3T3 CHO-K1 HEK293 Jurkat0
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Total ERK
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100HEK293NIH3T3
Cells x 10 3 / well
S/B
HTRF® assay are available for a large set of targets
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Phospho-protein Total protein
Phospho-Erk1/2 (Cellul’erk) Total ERK1/2
Phospho-AKT (Ser473)
Phospho-AKT (Thr308)
Phospho-JNK (Thr183/Tyr185)
Phospho-c Jun (Ser63)
Phospho-STAT3 (Tyr705) Total-STAT3
Phospho-p38 MAPK (Thr180/Tyr182)
Phospho-IKKb (Ser177/181)
Phospho-mTOR (Ser2448)
Phospho-CREB (Ser133)
Phospho-EGFR
Phospho-MEK1 (Ser218/222) Total-MEK1
Phospho-MEK1/2 (Ser218/222)
• Mix & Read solutions
• Faster & equally or more sensitive than othertechniques
• Cost efficient
• Amenable to all steps of drug research
Cisbio’s experts can design a custom assay for yourtarget of interest
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• Fast assay development
• More than 25 custom assays developed in 2012
• Success rate in 2012: 90%
Assay optimization
Assay transferand/or
implementationwith dedicated
expert
Confidentialityagreement
Paperstudy
Go / No Go
Assayfeasibilitystudy
Best pair analysis
Assay report
Reagent sourcing& labeling
Go / No Go
Data review
Custom kit supplies
Thank you