ULight-Acetyl-CoA Carboxylase (Ser79) Peptide and Europium-anti-phospho-Acetyl-CoA Carboxylase (Ser79) Antibody T E C H N I C A L AMPK␣1 Kinase Assay L A N C E U LT R A TECH NOTE U-TRF #12 Two LANCE Ultra companion products – two convenient sizes! POTENTIAL SUBSTRATE FOR KINASES: AMP-activated subfamily of protein kinases • TRF0118-D: 0.5 nmole, 1,000* assay points • TRF0118-M: 5 nmoles, 10,000* assay points *0.5 pmol/assay point PEPTIDE SEQUENCE: CHMRSAMSGLHLVKRR Synthetic peptide derived from residues 73-85 of rat acetylCoA carboxylase in which Ser77 is mutated to Ala; phosphorylation site: Ser79. Europium-anti-phospho-Acetyl-CoA Carboxylase (Ser79) Antibody: • TRF0208-D: 10 µg, 1,562* assay points • TRF0208-M: 100 µg, 15,625* assay points *40 fmol/assay point RECOGNIZED MOTIF: SSMpSGL VALIDATED FOR KINASE: AMPK␣1 Europium-labeled mouse monoclonal antibody recognizing phospho-Ser79 of rat acetyl-CoA carboxylase. 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 ULight-substrate phosphorylation. ␣1 Kinase Assay Development of an AMPK␣ Additional reagents AMPK␣1 active Carna # 02-113 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™-Acetyl-CoA Carboxylase (Ser79) Peptide (SAMS Peptide): Suggested procedure • Dilute the AMPK␣1 kinase, ATP, inhibitors and ULight-AcetylCoA Carboxylase peptide in Kinase Buffer. • Prepare a 4X Detection Mix by diluting the Eu-anti-phospho Acetyl-CoA Carboxylase antibody to 8 nM in 1X LANCE Detection Buffer. • Add to the wells of a white Optiplate-384: – 5 µL of AMPK␣1 enzyme – 2.5 µL of inhibitor or Kinase Buffer – 2.5 µL of ULight-Acetyl-CoA Carboxylase 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-Acetyl-CoA Carboxylase 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 Solution/Detection Mix to minimize the number of liquid handling steps. Experiment 1: Enzymatic Time Course Experiment 2: ATP Titration AMPK␣1 enzyme was incubated at concentrations ranging from 0.25 to 4 nM with 50 nM ULight-Acetyl-CoA Carboxylase 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 2 nM AMPK␣1 and 50 nM ULight-Acetyl-CoA Carboxylase 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 300 nM (final concentrations in 2% DMSO) were incubated with 2 nM AMPK␣1, 50 nM ULight-Acetyl-CoA Carboxylase peptide and 30 µM ATP. Kinase reactions were terminated after 30 min by the addition of EDTA. AMPK␣1 enzyme at 2 nM was incubated with 50 nM ULight-Acetyl-CoA Carboxylase peptide and 30 µM ATP with or without 1 µM staurosporine (final concentrations in 2% 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_02 Printed in USA