Using ULight - Poly GT (4:1) & Europium-Anti-Phospho-Tyrosine (PY20) Antibody Two LANCE® Ultra companion products — two convenient sizes! • TRF0100-D: 1 nmole, 1,000 assay points* • TRF0100-M: 10 nmoles, 10,000 assay points* *1 pmol/assay point • PEPTIDE SEQUENCE: [Glu-Tyr (4:1)] n • AD0066: 50 µg, 7,800 assay points* • AD0067: 1 mg, 150,000 assay points* *40 fmol/assay point • RECOGNIZED MOTIF: pTyr • Mouse monoclonal IgG2b antibody directed against phospho-tyrosine. Generic substrate for tyrosine kinases • VALIDATED FOR KINASES: p60c-src, AXL LANCE Ultra Kinase Assays • POTENTIAL SUBSTRATE FOR KINASES: most tyrosine kinases LANCE® Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W-1024 (Eu), with ULight, a new 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 phosphorylated ULight-labeled substrates brings donor and acceptor molecules into close proximity. After irradiation of the kinase reaction at 320 nm, the energy from the Eu donor is transferred to the ULight acceptor which, in turn, generates light at 665 nm. The intensity of the light emission is proportional to the level of ULight-substrate phosphorylation. Development of a p60c-src Kinase Assay Additional Reagents p60c-src, recombinant Upstate # 14-326 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 OptiPlate™-384, white PerkinElmer # 6007299 TopSeal-A™ PerkinElmer # 6005185 Kinase Buffer: 50 mM Tris-HCl, 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 ™-poly GT (4:1): Europium-anti-phospho-tyrosine (PY20) antibody: T E C H N I C A L LANCE Ultra p60c-src Kinase Assay L A N C E U LT R A T R - F R E T T E C H N O L O G Y TECH NOTE U-TRF #3 Suggested Procedure • Dilute kinase, ATP, inhibitors and ULight-poly GT (4:1) in Kinase Buffer. Experiment 1: Enzymatic Titration Assay Experiment 2: ATP Titration p60c-src enzyme at concentrations ranging from 0.3 pM to 30 nM was incubated with 100 nM ULight-poly GT (4:1) and 20 µM ATP. Kinase reactions were terminated after 60 min by the addition of EDTA. Serial dilutions of ATP ranging from 1 nM to 100 µM were added to 300 pM p60c-src kinase and 100 nM of ULight-poly GT (4:1) substrate. Kinase reactions were terminated after 60 min 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 2% DMSO) were pre-incubated for 5 min with the p60c-src enzyme (300 pM final concentration). Then 100 nM ULight-Poly GT (4:1) and 4 µM ATP were added. Kinase reactions were terminated after 60 min by the addition of EDTA. The p60c-src enzyme at 300 pM was incubated with 100 nM ULight-poly GT (4:1) substrate in Kinase Assay Buffer with 4 µM ATP or 10 µM staurosporine and ATP. Reactions were terminated after 60 min by the addition of EDTA. • Dilute antibody (Ab) in LANCE Detection Buffer to 8 nM. • Add to the wells of a white OptiPlate-384: 5 µL of p60 c-src enzyme, 2.5 µL of inhibitor or Kinase Buffer, 2.5 µL of ULight-poly GT (4:1)/ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). • Incubate the enzymatic reaction at room temperature (RT). • Stop the reaction by adding 5 µL of 40 mM EDTA in Detection Buffer. Leave 5 min at RT. • Add 5 µL of the antibody dilution (2 nM final concentration). • 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). Better p60c-src Kinase Assays with a Better Technology — LANCE Ultra For more information about LANCE Ultra, please visit www.perkinelmer.com/lanceultra or contact your local PerkinElmer Sales Representative. Learn more about our comprehensive range of reagents and consumables for drug discovery by visiting www.perkinelmer.com/ drugdiscovery. Complementary Products Product Description Product No. Europium-anti-phospho-Tyrosine (PT66) 50 µg 1 mg AD0068 AD0069 Europium-anti-phospho-Tyrosine (P-Tyr-100) 10 µg 50 µg 1 mg AD0203 AD0161 AD0162 ULight-poly GAT, [EAY(1:1:1)] n 1 nmole 10 nmoles TRF0101-D TRF0101-M PerkinElmer Life and Analytical Sciences 710 Bridgeport Avenue Shelton, CT 06484-4794 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 ©2006 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. LANCE is a registered trademark and EnVision, OptiPlate, TopSeal-A and ULight are trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. ULight is covered under patent number US2005202565 and US20040166515. 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. 007755_09 Printed in USA