Using ULight-Tyrosine Hydroxylase (Ser40) Peptide & Europium-anti-phospho-Tyrosine Hydroxylase (Ser40) Antibody ULight ™-Tyrosine Hydroxylase (Ser40) Peptide: • TRF0111-D: 0.5 nmole, 1,000 assay points* • TRF0111-M: 5 nmoles, 10,000 assay points* *0.5 pmol/assay point • CORE SEQUENCE MOTIF: RRQSLIE Synthetic peptide containing the residues surrounding Ser40 of tyrosine hydroxylase Phosphorylation site: Ser40 • VALIDATED FOR KINASES: PAK2, Aurora A, PKA, MSK1, CHK1 Europium-anti-phospho-Tyrosine Hydroxylase (Ser40) Antibody: • TRF0204-D: 10 µg, 1,562 assay points* • TRF0204-M: 100 µg, 15,625 assay points* *40 fmol/assay point • RECOGNIZED MOTIF: RRQpSLIE • Europium-labeled rabbit polyclonal antibody recognizing phospho-Ser40 in human tyrosine hydroxylase 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 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. Development of a PAK2 Kinase Assay Additional Reagents PAK2, active Upstate # 14-481 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 Two LANCE® Ultra companion products — two convenient sizes! T E C H N I C A L LANCE Ultra PAK2 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 #8 Suggested Procedure • Dilute kinase, ATP, inhibitors and ULight-Tyrosine Hydroxylase peptide in Kinase Buffer. Experiment 1: Enzymatic Time Course Experiment 2: ATP Titration PAK2 enzyme was incubated at concentrations ranging from 0.125 to 4 nM with 50 nM ULightTyrosine Hydroxylase 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 added to 0.5 nM PAK2 and 50 nM of ULight-Tyrosine Hydroxylase peptide. Kinase reactions were terminated after 30 min by the addition of EDTA. Experiment 3: Enzyme Inhibition Curve Experiment 4: Z'-factor Determination Serial dilutions of staurosporine ranging from 3 pM to 30 µM (final concentrations in 2% DMSO) were incubated with 0.5 nM PAK2, 50 nM ULight-Tyrosine Hydroxylase peptide and 10 µM ATP. Kinase reactions were terminated after 30 min by the addition of EDTA. PAK2 enzyme at 0.5 nM was incubated with 50 nM ULight-Tyrosine Hydroxylase peptide and 10 µM ATP with or without 10 µM staurosporine (final concentrations in 2% DMSO). Kinase reactions were terminated after 30 min by the addition of EDTA. • Prepare a 4X Detection Mix by diluting the Eu-anti-phosphoTyrosine Hydroxylase antibody to 8 nM in 1X LANCE Detection Buffer. • Add to the wells of a white OptiPlate-384: 5 µL of PAK2 enzyme, 2.5 µL of inhibitor or Kinase Buffer, 2.5 µL of ULight-Tyrosine Hydroxylase 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 the kinase reactions by adding 5 µL of 40 mM EDTA prepared in Detection Buffer. Leave for 5 min at RT. • Add 5 µL of Detection Mix (Euanti-phospho-Tyrosine Hydroxylase 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/Detection mix to minimize the number of liquid handling steps. Better PAK2 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/drug discovery. 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. 007778_04 Printed in USA