T e c h n i c a l N o t e U-TRF #35 LANCE Ultra SET7/9 Histone H3-Lysine N-methyltransferase Assay Authors Nancy Gauthier Valérie Paquet Liliana Pedro Anne Labonté Anja Rodenbrock Marjolaine Roy Lucille Beaudet Roberto Rodriguez-Suarez LANCE® Ultra PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 This LANCE® Ultra immunodetection assay measures the mono-methylation of a biotinylated Histone H3 (1-21) peptide at lysine 4. K B + Enzyme Europium-anti-methyl-Histone H3 Lysine 4 (H3K4me1-2) Antibody • TRF0402-D: 10 µg, 1,562 assay points* • TRF0402-M: 100 µg, 15,625 assay points* *40 fmol/assay point SAM SAH Me K B Peptidic Substrate Sequence: ARTKQTARKSTGGKAPRKQLA-GG-K(BIOTIN)-NH2 + 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 (SA) which bring 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 Me ULight B K Figure 1. Schematic representation of the LANCE Ultra detection of a modified histone peptide. Development of a SET7/9 Histone H3-Lysine N-methyltransferase Assay Reagents needed for the assay: Europium-anti-methyl-Histone H3 Lysine 4 (H3K4me1-2) LANCE Ultra ULight-Streptavidin Histone H3 (1-21) peptide, biotinylated LANCE Detection Buffer, 10X SET7/9 (human), recombinant White opaque OptiPlate™-384 TopSeal™-A films S-(5'-Adenosyl)-L-methionine chloride (SAM) Sinefungin S-(5'-Adenosyl)-L-homocysteine (SAH) PerkinElmer # TRF0402 PerkinElmer # TRF0102 AnaSpec # 61702 PerkinElmer # CR97-100 Enzo # ALX-201-178-C100 PerkinElmer # 6007299 PerkinElmer # 6005185 Sigma # A7007 Sigma # S8559 Sigma # A9384 Standard Protocol • Dilute SET7/9 enzyme, SAM, inhibitors and biotinylated peptide substrate in Assay Buffer just before use. • Add to the wells of a white Optiplate-384: – 5 μL of inhibitor (2X) or Assay Buffer – 2.5 μL of enzyme (4X) – 2.5 μL of biotinylated Histone H3 (1-21) peptide/SAM mix (4X). For SAM titration, add SAM dilutions independently of substrate. • Cover the plate with TopSeal-A film and incubate at room temperature (RT). • Prepare Detection Mix* by diluting the Eu-Ab to 4 nM and ULight-Streptavidin to 100 nM in 1X LANCE Detection Buffer (final concentrations of 2 nM and 50 nM respectively, in 20 µL total assay volume). • Add 10 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 EnVision® Multilabel Reader in TR-FRET mode (excitation at 320 or 340 nm & emission at 665 nm). Experiment 1: Enzyme Titration and Time-Course Experiment 3: Enzyme Inhibition 70,000 [SET7/9] (nM) 60,000 10 5 2.5 1 0.5 0 50,000 40,000 30,000 20,000 10,000 0 LANCE Signal (665 nm) * Under these experimental conditions, addition of the Detection Mix stops SET7/9 enzymatic reaction. LANCE Signal (665 nm) SAM is prepared at 30 mM in 5 mM H2SO4/10% ethanol (v/v) in H2O, aliquoted and stored at -80 °C. Assay Buffer: 50 mM Tris-HCl, pH 8.8, 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20 60,000 Sinefungin IC50 = 769 nM 50,000 40,000 SAH IC50 = 93 μM 30,000 20,000 10,000 0 10 20 30 40 50 60 70 80 90 100 0 -∞ -9 -8 -7 -6 -5 -4 -3 Log [Inhibitor] (M) Time (min) Experiment 2: SAM Titration Experiment 4: Z’-factor Determination 70,000 60,000 LANCE Signal (665 nm) Serial dilutions of sinefungin ranging from 1 nM to 1 mM and SAH ranging from 10 nM to 1 mM were pre-incubated for 10 min with 5 nM SET7/9. Enzymatic reactions were initiated by the addition of 200 nM biotinylated H3 (1-21) peptide substrate plus 300 nM SAM. Enzymatic reactions contain 1% DMSO. LANCE Signal (665 nm) Enzymatic progress curves were performed by incubating SET7/9 at concentrations ranging from 0.5 to 10 nM with 200 nM biotinylated H3 (1-21) peptide substrate and 300 μM SAM. Detection Mix was added to stop the reactions at the indicated times and signal was read after 60 min. A 60 min reaction time using 5 nM enzyme was selected for all subsequent experiments. Km app = 75 nM 50,000 40,000 30,000 20,000 10,000 0 -∞ -11 -10 -9 -8 -7 -6 -5 -4 Log [SAM] (M) Serial dilutions of SAM ranging from 30 pM to 100 µM were added to 5 nM SET7/9 and 200 nM biotinylated H3 (1-21) peptide substrate. A 300 nM SAM concentration was selected for subsequent experiments. 60,000 No inhibitor 50,000 40,000 30,000 20,000 300 µM Sinefungin 10,000 0 0 10 20 Copyright © 2010, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. Oct. 2010 40 50 SET7/9 (5 nM) was pre-incubated with or without 300 µM sinefungin for 10 min. Enzymatic reactions were initiated by the addition of 200 nM biotinylated H3 (1-21) peptide substrate plus 300 nM SAM. Enzymatic reactions contain 1% DMSO. For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs Printed in USA 30 Well # PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com 009371_01 Z' = 0.88 S/B = 9.1