T E C H N I C A L N O T E

mTOR Kinase AssayULight™ -eIF4E-binding protein 1 (Thr37/46) Peptide Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) Antibody

ULight™ -4E-BP1 (Thr37/46) Peptide:• TRF0128-D: 0.5 nmole, 1,000 assay points*• TRF0128-M: 5 nmoles, 10,000 assay points**0.5 pmol/assay point

PEPTIDE MOTIF:STTPGGTLFSTTPGSynthetic peptide containing the residues surrounding Thr37 and Thr46 of human eukaryotic translation initiation factor 4E-binding protein 1; phosphorylation sites: Thr37 and Thr46.

Europium-anti-phospho-4E-BP1(Thr37/46) Antibody:• TRF0216-D: 10 µg, 1,562 assay points*• TRF0216-M: 100 µg, 15,625 assay points**40 fmol/assay point

RECOGNIZED MOTIF: Europium-labeled rabbit monoclonal antibody recognizing human eukaryotic translation initiation factor 4E-binding protein 1 phosphorylated at Thr 37 or Thr 46.

LANCE Ultra(ULight-labeled substrate)

Kinase +

+ Eu-Ab

Eu-Ab

ULight

ULight

P

PULight

Ex 320 or 340 nmEm 665 nm

FRET

LANCE® Ultra

TECH NOTE U-TRF #29

Two LANCE® Ultra companion products – two convenient sizes!

LANCE ULTRA KINASE ASSAYSLANCE Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye with ULight, an innovative small molecular weight acceptor dye with a red-shifted fluorescent emission. In kinase assays, the binding of an Eu-labeled anti-phospho-substrate antibody to the phosphorylated ULight-labeled substrate brings donor and acceptor molecules into close proximity.

After irradiation of the kinase reaction at 320 or 340 nm, the energy from the Eu donor is transferred to the ULight acceptor dye which, in turn, generates light at 665 nm. The intensity of the light emission is proportional to the level of ULight-substrate phosphorylation.

Authors

Mireille CaronHendrick PlanteMarie-Claude LoiselleThomas LassalleValérie PaquetLucille Beaudet Roberto Rodriguez-Suarez PerkinElmer Inc.940 Winter StreetWaltham, MA 02451USA

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Copyright ©2009, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 008666_01 Printed in USA

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com

Development of a mTOR Kinase AssayAdditional Reagents:

LANCE® Detection Buffer, 10X PerkinElmer # CR97-100OptiPlate™-384, white PerkinElmer # 6007299TopSeal™-A PerkinElmer # 6005185mTOR Millipore # 14-770Kinase Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20.

Note: MnCl2 might not be required for other kinases.

Suggested Procedure

• Dilutethe mTOR enzyme, ATP, inhibitors and ULight-4E-BP1 Peptide in Kinase Buffer.

• Preparea4XDetectionMixbydilutingtheEu-anti-phospho-4E-BP1Antibody to 8 nM in 1X LANCE Detection Buffer.

• AddtothewellsofawhiteOptiplate-384:– 5μLofmTORenzyme– 2.5μLofinhibitororKinaseBuffer– 2.5μLofULight-4E-BP1 (Thr37/46) Peptide/ATP mix

(for ATP titration, ATP dilutions are added separately in Kinase Buffer).• CovertheplatewithTopSeal-Aandincubateatroomtemperature(RT).• Stopkinasereactionsbyadding5μLof32mMEDTApreparedin1X

Detection Buffer (Stop Solution). Leave for 5 min at RT.• Add5μLofDetectionMix(Eu-anti-phospho-4E-BP1(Thr37/46)Antibody

at a final concentration of 2 nM).• CoverwithTopSeal-Aandincubatefor1hatRT.• RemoveTopSeal-AandreadsignalwiththeEnVision® Multilabel Reader

in TR-FRET mode (excitation at 320 nm & emission at 665 nm).

NOTE: Eu-labeled antibodies and EDTA can be premixed just before use as a 2X concentrated Stop Solution/Detection mix to minimize the number of liquid handling steps.

Experiment 1: Enzyme Titration and Time-Course

mTOR enzyme was incubated at concentrations ranging from 0.1 to 2 nM with 50 nM ULight-4E-BP1 (Thr37/46) Peptide and 100 μM ATP. Kinase reactions were terminated after 0 to 120 min by the addition of EDTA.

Experiment 4: Z’-factor Determination

mTOR enzyme at 0.5 nM was incubated with 50 nM ULight-4E-BP1 Peptide and 10 μM ATP with or without 30 μM PI-103 (final concentrations in 1% DMSO). Kinase reactions were terminated after 60 min by the addition of EDTA.

0 20 40 60 80 100 120 1400

20,000

40,000

60,000

80,000

100,000

120,000210.50.250.10

[mTOR] (nM)

Time (min)

LA

NC

E S

ign

al (

665

nm

)

Experiment 2: ATP Titration

Serial dilutions of ATP ranging from 10 nM to 1 mM were added to 0.5 nM mTOR enzyme and 50 nM of ULight-4E-BP1 (Thr37/46) Peptide. Kinase reactions were terminated after 60 min by the addition of EDTA.

0

20,000

40,000

60,000

80,000

-8 -7 -6 -5 -4 -3 -2-∞

Km app = 10.8 µM

Log [ATP] (M)

LA

NC

E S

ign

al (

665

nm

)

0 5 10 15 20 250

5,000

10,000

15,000

20,000No PI-103

+ 30 µM PI-103

Z' = 0.88

Well #

LA

NC

E S

ign

al (

665

nm

)

Experiment 3: Enzyme Inhibition

Serial dilutions of PI-103 ranging from 0.3 nM to 30 μM (final concentrations in 1% DMSO) were incubated with 0.5 nM mTOR enzyme, 50 nM ULight-4E-BP1 Peptide and 10 μM ATP. Kinase reactions were terminated after 60 min by the addition of EDTA.

0

5,000

10,000

15,000

20,000

-10 -9 -8 -7 -6 -5 -4-∞

IC50 = 27 nM

Log [PI-103] (M)

LA

NC

E S

ign

al (

665

nm

)