ULight-mTOR (Ser2448) Peptide and Europium-anti-phospho-mTOR (Ser2448) Antibody T E C H N I C A L p70 S6K Kinase Assay L A N C E U LT R A TECH NOTE U-TRF #13 Two LANCE Ultra companion products – two convenient sizes! • TRF0119-D: 0.5 nmole, 1,000 assay points* Europium-anti-phospho-mTOR (Ser2448) Antibody: • TRF0119-M: 5 nmoles, 10,000 assay points* • TRF0209-D: 10 µg, 1,562 assay points* *0.5 pmol/assay point • TRF0209-M: 100 µg, 15,625 assay points* PEPTIDE SEQUENCE: *40 fmol/assay point CAGAGRSRTRTDSYSAGQSV RECOGNIZED MOTIF: Synthetic peptide containing the residues surrounding Ser2448 of human mTOR; phosphorylation site: Ser2448. RSRTRTDpSYSAGQSV VALIDATED FOR KINASES: p70 S6K, MST1, IRAK4, JNK1, JNK2, GSK-3B, MLK3 Europium-labeled rabbit monoclonal antibody recognizing phospho-Ser2448 in human mTOR. LANCE Ultra Kinase Assays LANCE® Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W-1024 (Eu), with ULight, an innovative small molecular weight acceptor dye with a red-shifted fluorescent emission. In kinase assays, the binding of an Eu-labeled antiphospho-substrate antibody to the phosphorylated ULightlabeled 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 ULightsubstrate phosphorylation. Development of a p70 S6K Kinase Assay Additional reagents p70 S6 Kinase, active Upstate # 14-486 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 OptiPlate™-384, white PerkinElmer # 6007299 TopSeal™-A PerkinElmer # 6005185 Kinase Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20. w w w. p e r k i n e l m e r. c o m N O T E ULight™-mTOR (Ser2448) Peptide: Suggested procedure • Dilute the p70 S6K enzyme, ATP, inhibitors and ULight-mTOR peptide in Kinase Buffer. • Prepare a 4X Detection Mix by diluting the Eu-anti-phosphomTOR antibody to 8 nM in 1X LANCE Detection Buffer. • Add to the wells of a white Optiplate-384: – 5 µL of p70 S6K enzyme – 2.5 µL of inhibitor or Kinase Buffer – 2.5 µL of ULight-mTOR peptide/ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). • Stop kinase reactions by adding 5 µL of 40 mM EDTA prepared in 1X Detection Buffer (Stop Solution). Leave for 5 min at RT. • Add 5 µL of 4X Detection Mix (Eu-anti-phospho-mTOR antibody at a final concentration of 2 nM). • Cover with TopSeal-A and incubate for 1 h at RT. • Remove TopSeal-A and read signal with the EnVision® Multilabel Reader in TR-FRET mode (excitation at 320 nm and emission at 665 nm). NOTE: Eu-labeled antibodies and EDTA can be premixed before use as a 2X concentrated Stop Solution/Detection Mix to minimize the number of liquid handling steps. • Cover the plate with TopSeal-A and incubate at room temperature (RT). Experiment 1: Enzymatic Time Course Experiment 2: ATP Titration p70 S6K enzyme was incubated at concentrations ranging from 0.5 to 8 nM with 50 nM ULight-mTOR peptide and 20 µM ATP. Kinase reactions were terminated after 0 to 120 min by the addition of EDTA. Serial dilutions of ATP ranging from 10 nM to 1 mM were incubated with 2 nM p70 S6K enzyme and 50 nM of ULight-mTOR peptide. Kinase reactions were terminated after 45 min by the addition of EDTA. Experiment 3: Enzyme Inhibition Curve Experiment 4: Z’-factor Determination Serial dilutions of staurosporine ranging from 30 pM to 30 µM (final concentrations in 2% DMSO) were incubated with 2 nM p70 S6K enzyme, 50 nM ULight-mTOR peptide and 3 µM ATP. Kinase reactions were terminated after 45 min by the addition of EDTA. p70 S6K enzyme at 2 nM was incubated with 50 nM ULight-mTOR peptide and 3 µM ATP with or without 1 µM staurosporine (final concentrations in 2% DMSO). Kinase reactions were terminated after 45 min by the addition of EDTA. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices ©2007 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. OptiPlate, TopSeal and ULight are trademarks and EnVision, and LANCE are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors. 007870_01 Printed in USA