T E C H N I C A L N O T E U-TRF #17 LANCE Ultra c-TAK1 Kinase Assay PerkinElmer, Inc. Montreal, QC Canada H3J 1R4 LANCE® Ultra TR-FRET Technology This LANCE Ultra kinase assay measures the phosphorylation of a Cdc25C peptide substrate at Ser216. ULight™-Cdc25C (Ser216) Peptide: • TRF0123-D: 1 nmole, 1,000* assay points LANCE Ultra (ULight-labeled substrate) ULight • TRF0123-M: 10 nmoles, 10,000* assay points Kinase + *1 pmol/assay point Europium-anti-phospho-(Ser) 14-3-3 binding motif 4E2 Antibody: • AD0192: 10 µg, 1,562* assay points P ULight + Eu-Ab *40 fmol/assay point Recognized Motif: Eu FRET Europium-labeled mouse monoclonal antibody. Peptide Sequence: Ex 320 or 340 nm Em 665 nm (R/K)XXpSXP Eu-Ab P ULight CSRSGLYRSPSMPENLNRPRL Synthetic peptide derived from residues 207-226 of human M-phase inducer phosphatase 3 (Cdc25C); phosphorylation site: Ser216. LANCE Ultra Kinase Assays: LANCE Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assays use a proprietary europium chelate donor dye, W1024 (Eu), together with ULight™, a small molecular weight acceptor dye with a red-shifted fluorescent emission. In this technical note, we present the optimization of a c-TAK1 kinase assay using a ULightlabeled peptide substrate. The binding of the Eu-labeled anti-phospho-(Ser) 14-3-3 binding motif antibody to the Cdc25C peptide substrate at Ser216 brings the Eu donor and ULight acceptor dye molecules into close proximity. Upon irradiation 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. Figure 1. Schematic representation of the LANCE Ultra detection of a phosphorylated peptide substrate. Development of a c-TAK1 Kinase Assay Reagents needed for this assay: Europium-labeled anti-phospho-(Ser) 14-3-3 binding motif 4E2 Antibody PerkinElmer # AD0192 ULight-Cdc25C (Ser216) PerkinElmer # TRF0123 C-TAK1 active Millipore # 14-375 LANCE® Detection Buffer, 10X PerkinElmer # CR97-100 ™ OptiPlate -384, white PerkinElmer # 6007299 TopSeal™-A film PerkinElmer # 6050195 Kinase Buffer: 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20 Standard Protocol • Dilute the c-TAK1 kinase, ATP, inhibitors and ULight-Cdc25C (Ser216) peptide in Kinase Buffer. • Prepare a 4X Detection Mix by diluting the Eu-anti-phospho-(Ser) 14-33 binding motif 4E2 antibody to 8 nM in 1X LANCE Detection Buffer. • Add to the wells of a white OptiPlate-384: – 5 μL of c-TAK1 enzyme – 2.5 μL of inhibitor or Kinase Buffer – 2.5 μL of ULight-Cdc25C (Ser216) peptide/ATP mix (for ATP titration, ATP dilutions are added separately in Kinase Buffer). • Cover the plate with TopSeal-A film and incubate at room temperature (RT). • Stop kinase reactions by adding 5 μL of 40 mM EDTA prepared in 1X LANCE Detection Buffer (Stop Solution). Leave for 5 min at RT. • Add 5 μL of 4X Detection Mix (Eu-anti-phospho-(Ser) 14-3-3 binding motif 4E2 antibody at a final concentration of 2 nM). • Cover with TopSeal-A film and incubate for 1 h at RT. • Remove the TopSeal-A film and read signal with the EnVision® Multilabel Reader in TR-FRET mode (excitation at 320 or 340 nm and emission at 665 nm). NOTE: Eu-labeled antibodies and EDTA can be premixed just before use as a 2X concentrated Stop Solution/Detection Mix to minimize the number of liquid handling steps. Experiment 1: Enzymatic Titration and Time Course Experiment 2: ATP Titration Enzymatic progress curves were produced by incubating c-TAK1 enzyme at concentrations ranging from 1.25 to 10 nM with 100 nM ULight-Cdc25C (Ser216) peptide and 50 µM ATP. Kinase reactions were terminated at the indicated times by the addition of EDTA. Detection mix was added and signal read after 60 minutes. Serial dilutions of ATP ranging from 10 nM to 1 mM were added to 5 nM c-TAK1 and 100 nM ULight-Cdc25C (Ser216) peptide. Kinase reactions were terminated after 90 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 300 nM (final concentrations in 2% DMSO) were incubated with 5 nM c-TAK1, 100 nM ULight-Cdc25C (Ser216) peptide and 100 µM ATP. Kinase reactions were terminated after 45 min by the addition of EDTA. c-TAK1 enzyme at 5 nM was incubated with 100 nM ULight-Cdc25C (Ser216) peptide in kinase assay buffer with 100 µM ATP, and 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 P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Copyright ©2007-2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 010232_01 Printed in USA Apr. 2012