T e c h n i c a l N o t e U-TRF #46 LANCE Ultra HDAC2 Histone H3 Lysine 4 Deacetylase Assay Authors Julie Blouin Mireille Caron Lucille Beaudet PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 LANCE® Ultra This LANCE Ultra immunodetection assay measures the deacetylation of a biotinylated Histone H3 (1-21) peptide acetylated at lysine 4. Ac K B Europium-anti-unmodified Histone H3 Lysine 4 (H3K4) Antibody + Enzyme • TRF0404-D: 10 µg, 1,562 assay points* • TRF0404-M: 100 µg, 15,625 assay points* *40 fmol/assay point K B Peptidic Substrate Sequence: + Eu-Ab + ULight-SA ARTK(ac)QTARKSTGGKAPRKQLA-GG-K(BIOTIN)-OH 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. In this technical note, we present the optimization of an epigenetic enzymatic assay using a biotinylated Histone H3-derived peptide as substrate. The deacetylated peptide product is captured by the Eu-labeled antibody (Eu-Ab) and ULight-Streptavidin (ULight-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. Eu FRET Emission 665 nm ULight B Excitation 320 or 340 nm K Figure 1. Schematic representation of the LANCE Ultra detection of a deacetylated histone peptide. Development of a HDAC2 Histone H3-Lysine 4 Deacetylase Assay: Reagents needed for the assay: Europium-anti-unmodified Histone H3 Lysine 4 (H3K4) Antibody PerkinElmer # TRF0404 LANCE Ultra ULight-Streptavidin PerkinElmer # TRF0102 Histone H3 (1-21), H3K4ac peptide, biotinylated AnaSpec # 65207 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 HDAC2 (human), recombinant BPS BioScience # 50002 Trichostatin A Sigma # T-8552 SAHA Cayman Chemical # 10009929 White opaque OptiPlate -384 PerkinElmer # 6007299 TopSeal -A films PerkinElmer # 6005185 ™ ™ Assay Buffer: 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DDT, 0.01% Tween-20 and 0.01% BSA. Standard Protocol • Dilute HDAC2 enzyme, inhibitors and biotinylated Histone H3K4ac 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 • Incubate 5 min at room temperature (RT). – 5 μL of biotinylated Histone H3K4ac peptide (2X) • Cover the plate with TopSeal-A film and incubate at RT • Prepare a 4X Stop Solution containing 12 µM of Trichostatin A in 1X LANCE Detection Buffer (final concentration of 3 µM Trichostatin A in 20 µL total assay volume). • 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). Add to the wells: – 5 μL of Trichostatin A Stop Solution. 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). Experiment 1: Enzyme Titration and Time-Course Experiment 2: Enzyme Inhibition Enzymatic progress curves were performed by incubating HDAC2 at concentrations ranging from 2.5 to 20 nM with 1 µM biotinylated Histone H3K4ac peptide substrate. Reactions were stopped by the addition of Trichostatin A at indicated times. Detection mix was then added and signal read after 60 min. A 60 min reaction time using 5 nM enzyme was selected for all subsequent experiments. Serial dilutions of Trichostatin A ranging from 10 pM to 100 µM and serial dilutions of SAHA ranging from 100 pM to 1 mM were pre-incubated for 5 min with 5 nM of HDAC2. Enzymatic reactions were initiated by the addition of 1 µM biotinylated Histone H3K4ac peptide substrate. Enzymatic reactions contain 2% DMSO. Experiment 3: Z'-factor Determination HDAC2 (5 nM) was pre-incubated with or without 3 µM Trichostatin A for 5 min. Enzymatic reactions were initiated by the addition of 1 µM biotinylated Histone H3K4ac peptide substrate. Enzymatic reactions contain 2% 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 ©2011, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 009814_01 Printed in USA Aug. 2011