ULight-PKC Peptide and Europium-anti-phospho-PKC (Ala25Ser) Peptide Antibody Two LANCE Ultra companion products – two convenient sizes! • TRF0108-D: 0.5 nmole, 1,000* assay points Europium-anti-phospho-PKC (Ala25Ser) Peptide Antibody: • TRF0108-M: 5 nmoles, 10,000* assay points • TRF0207-D: 10 µg, 1,562* assay points *0.5 pmol/assay point • TRF0207-M: 100 µg, 15,625* assay points PEPTIDE SEQUENCE: *40 fmol/assay point CRFARKGSLRQKNV Synthetic peptide derived from amino acids 19-31 of PKC␣; phosphorylation site: Ser25. VALIDATED FOR KINASES: PKC␣, PKC1, PKC2, PKC␦, PKC⑀, PKC␥, PKC, PKC, PKC, PKC RECOGNIZED MOTIF: RFARKGpSLRQKNV Europium-labeled mouse monoclonal antibody recognizing phospho-Ser25 in human PKC in which Ala25 is mutated to Ser and phosphorylated. 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 a PKC Kinase Assay Additional reagents PKC-␣ Invitrogen # P2227 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™-PKC peptide: T E C H N I C A L PKC Kinase Assay L A N C E U LT R A TECH NOTE U-TRF #11 Suggested procedure • Dilute the PKC-␣ kinase, ATP, inhibitors and ULight-PKC Peptide 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. • Prepare a 4X Detection Mix by diluting the Eu-anti-phospho PKC (Ala25Ser) antibody to 8 nM in 1X LANCE Detection Buffer. • Add 5 µL of 4X Detection Mix (Eu-anti-phospho-PKC (Ala25Ser) Antibody at a final concentration of 2 nM). • Add to the wells of a white OptiPlate-384: – 5 µL of PKC-␣ enzyme – 2.5 µL of inhibitor or Kinase Buffer – 2.5 µL of ULight-PKC substrate/ ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). • Cover with TopSeal-A and incubate for 1 h at RT. • Cover the plate with TopSeal-A and incubate for 30 min at room temperature (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 PKC-␣ enzyme was incubated at concentrations ranging from 1 to 100 pM with 50 nM ULight-PKC substrate and 10 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 10 pM PKC-␣ and 50 nM ULight-PKC substrate. 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 30 pM to 3 µM (final concentrations in 1% DMSO) were incubated with 10 pM PKC-␣, 50 nM ULight-PKC substrate and 10 µM ATP. Kinase reactions were terminated after 30 min by the addition of EDTA. PKC-␣ enzyme at 10 pM was incubated with 50 nM ULight-PKC substrate and 10 µM ATP with or without 1 µM staurosporine (final concentrations in 1% DMSO). Kinase reactions were terminated after 30 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_03 Printed in USA