ULight-IRS-1 (Tyr983) Peptide and Europium-anti-phospho-tyrosine Antibody (PT66) Two LANCE Ultra companion products – two convenient sizes! ULight™-IRS-1 (Tyr983) Peptide: • TRF0120-M: 10 nmoles, 10,000* assay points • AD0068: 50 µg, 7,500* assay points *1 pmol/assay point • AD0069: 1 mg, 150,000* assay points PEPTIDE SEQUENCE: *40 fmol/assay point CKKSRGDYMTMQIG RECOGNIZED MOTIF: Synthetic peptide derived from residues 979-989 of mouse Insulin receptor substrate 1 (IRS-1); phosphorylation site: Tyr983. pTyr VALIDATED FOR KINASES: INSR, EphB4, FGFR1, FLT1, IGFR1, JAK1, JAK2, PDGFRa, RET, TIE2, TYK2 Mouse monoclonal antibody directed against phosphotyrosine. POTENTIAL SUBSTRATE FOR KINASES: Tyrosine kinases specific for the YMxM motif 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 a 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 an INSR Kinase Assay Additional reagents INSR active Carna # 08-242 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 • TRF0120-D: 1 nmole, 1,000* assay points Europium-anti-phospho-tyrosine Antibody (PT66): T E C H N I C A L INSR Kinase Assay L A N C E U LT R A TECH NOTE U-TRF #14 Suggested procedure • Dilute the INSR kinase, ATP, inhibitors and ULight-IRS-1 (Tyr983) peptide in Kinase Buffer. • Prepare a 4X Detection Mix by diluting the Eu-anti-phosphotyrosine antibody (PT66) to 8 nM in 1X LANCE Detection Buffer. • Add to the wells of a white Optiplate-384: – 5 µL of INSR enzyme – 2.5 µL of inhibitor or Kinase Buffer – 2.5 µL of ULight-IRS-1 (Tyr983) peptide/ ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). • Cover the plate with TopSeal-A and incubate at room temperature (RT). • 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–tyrosine Antibody (PT66) 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. Experiment 1: Enzymatic Time Course Experiment 2: ATP Titration INSR enzyme was incubated at concentrations ranging from 2.5 to 20 nM with 100 nM ULight-IRS-1 (Tyr983) peptide and 1 mM ATP. Kinase reactions were terminated after 0 to 240 min by the addition of EDTA. Serial dilutions of ATP ranging from 10 nM to 1 mM were added to 10 nM INSR and 100 nM ULight-IRS-1 (Tyr983) peptide. Kinase reactions were terminated after 4 hours by the addition of EDTA. Experiment 3: Enzyme Inhibition Curve Experiment 4: Z’-factor Determination Serial dilutions of staurosporine ranging from 100 pM to 10 µM (final concentrations in 1% DMSO) were incubated with 10 nM INSR, 100 nM ULight-IRS-1 (Tyr983) and 1 mM ATP. Kinase reactions were terminated after 4 hours by the addition of EDTA. INSR enzyme at 10 nM was incubated with 100 nM ULight-IRS-1 (Tyr983) peptide and 1 mM ATP with or without 10 µM staurosporine (final concentrations in 1% DMSO). Kinase reactions were terminated after 4 hours by the addition of EDTA. These results show that LANCE Ultra reagents can be used to monitor the activity of kinases such as INSR with very high Km for ATP. 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_07 Printed in USA