T e c h n i c a l N o t e U-TRF #40 LANCE Ultra EZH2 Histone H3-Lysine 27 N-methyltransferase Assay Authors Julie Blouin Mathieu Arcand Mireille Caron Anne Labonté Claire Normand Lucille Beaudet Jaime Padrós LANCE® Ultra PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 This LANCE Ultra immunodetection assay measures the dimethylation of a biotinylated Histone H3 (21-44) peptide at lysine 27. K B + Enzyme Europium-anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) Antibody • TRF0406-D: 10 µg, 1,562 assay points* • TRF0406-M: 100 µg, 15,625 assay points* *40 fmol/assay point SAM SAH MeMe K B Peptidic Substrate Sequence: ATKAARKSAPATGGVKKPHRYRP-GG-K(Biotin)-OH + Eu-Ab + ULight-SA LANCE Ultra 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. Eu In this technical note, we present the optimization of an epigenetic enzymatic assay using a biotinylated Histone H3-derived peptide as substrate. The modified peptide is captured by the Eu-labeled antibody (Eu-Ab) and ULight-Streptavidin (ULight-SA), which 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 biotinylated substrate modification. FRET Em 665 nm Excitation 320 or 340 nm MeMe ULight B K Figure 1. Schematic representation of the LANCE Ultra detection of a modified histone peptide. Development of an EZH2 Histone H3-Lysine 27 N-methyltransferase Assay: Reagents needed for the assay: Europium-anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) Antibody PerkinElmer # TRF0406 LANCE Ultra ULight-Streptavidin PerkinElmer # TRF0102 Histone H3 (21-44) peptide, biotinylated AnaSpec # 64440 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 EZH2/EED/SUZ12/RbAp48/AEBP2 Complex BPS BioScience # 51004 White opaque OptiPlate™-384 PerkinElmer # 6007299 TopSeal™-A films PerkinElmer # 6005185 Sinefungin Sigma # S8559 S-(5’-Adenosyl)-L-methionine chloride (SAM) Sigma # A7007 Poly-L-lysine solution – 0.1% (w/v) Sigma # P8920 SAM is prepared at 30 mM in 5 mM H2SO4/10% ethanol (v/v) in H2O, aliquoted and stored at -80 ºC. Assay Buffer: 50 mM Tris-HCl pH 9.0, 50 mM NaCl, 1 mM DTT, 0.01% Tween-20 and 0.01% BSA. 60,000 Experiment 3: Enzyme Inhibition [EZH2] (ng/well) 50,000 200 150 100 75 0 40,000 30,000 20,000 10,000 0 0 30 60 90 120 Time (min) 150 LANCE Signal (665 nm) LANCE Signal (665 nm) Experiment 1: Enzyme Titration and Time-Course Standard Protocol • Dilute EZH2 enzyme complex, SAM, sinefungin (inhibitor) and biotinylated peptide substrate in Assay Buffer just before use. • Add to the wells of a white OptiPlate-384: – 2.5 μL of enzyme (4X) – 2.5 μL of inhibitor (4X) or Assay Buffer – 5 μL of biotinylated Histone H3 (21-44) peptide/SAM Mix (2X). For SAM titration, add SAM dilutions independently of substrate. • Cover the plate with TopSeal-A film and incubate at room temperature (RT). • Prepare a 4X Detection Mix by diluting the Eu-Ab to 8 nM and ULightStreptavidin to 200 nM in 1X LANCE Detection Buffer (final concentrations of 2 nM and 50 nM, respectively, in 20 µL total assay volume). • Prepare a 4X Stop Solution containing 0.0004% of poly-L-lysine in 1X LANCE Detection Buffer (final concentration of 0.0001% in 20 µL total assay volume). – 5 μL of poly-L-lysine Stop Solution and incubate 5 min at RT – 5 μL of Detection Mix • Cover with TopSeal-A film and incubate for 60 min 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 & emission at 665 nm). 180 30,000 IC50: 780 μM 25,000 20,000 15,000 10,000 5,000 0 -∞ -7 -6 -5 -4 -3 -2 -1 Log [Sinefungin] (M) Experiment 2: SAM Titration Experiment 4: Z’-factor Determination 50,000 40,000 LANCE Signal (665 nm) Serial dilutions of sinefungin ranging from 300 nM to 30 mM were pre-incubated for 10 min with 150 ng/well EZH2 complex. Enzymatic reactions were initiated by the addition of 500 nM biotinylated Histone H3 (21-44) peptide substrate plus 3 µM SAM. Enzymatic reactions contain 1% DMSO. LANCE Signal (665 nm) Enzymatic progress curves were performed by incubating EZH2 complex at concentrations ranging from 75 to 200 ng/well with 500 nM biotinylated Histone H3 (21-44) peptide substrate and 30 µM SAM. Reactions were stopped by the addition of poly-L-lysine at indicated times. The Detection Mix was then added and signal was read after 60 min. A 180 min reaction time using 150 ng/well enzyme was selected for all subsequent experiments. Km app = 1.3 μM 30,000 20,000 10,000 0 -∞ -9 -8 -7 -6 -5 Log [SAM] (M) -4 -3 Serial dilutions of SAM ranging from 3 nM to 300 µM were added to 150 ng/well EZH2 complex and 500 nM biotinylated Histone H3 (21-44) peptide substrate. A 3 µM SAM concentration was selected for subsequent experiments. 30,000 No inhibitor 25,000 20,000 15,000 10,000 10 mM Sinefungin 5,000 0 0 5 10 15 20 25 30 35 40 45 50 Well # EZH2 complex (150 ng/well) was pre-incubated with or without 10 mM sinefungin for 10 min. Enzymatic reactions were initiated by the addition of 500 nM biotinylated Histone H3 (21-44) peptide substrate plus 3 µM SAM. Enzymatic reactions contain 1% DMSO. 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 © 2010-2011, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 009570_01 Printed in USA Mar. 2011 Z' = 0.8 S/B = 4.6