T E C H N I C A L N O T E AlphaLISA #6 AlphaLISA HDAC1 Histone H3-Lysine 27 Deacetylase Assay Authors Julie Blouin Mathieu Arcand Mireille Caron Anne Labonté Claire Normand Lucille Beaudet Jaime Padrós AlphaLISA® PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 This AlphaLISA immunodetection assay measures the deacetylation of a biotinylated Histone H3 (21-44) peptide acetylated at lysine 27. Anti-acetyl Histone H3 Lysine 27 (H3K27ac) AlphaLISA® Acceptor Beads • AL120C: 250 µg, 500 assay points* • AL120M: 5 mg, 10,000 assay points* • AL120R: 25 mg, 50,000 assay points* *0.5 µg/assay point Peptidic Substrate Sequence: ATKAARK(ac)SAPATGGVKKPHRYRP-GG-K(Biotin)-OH AlphaLISA Assays AlphaLISA technology is a powerful and versatile platform that offers highly sensitive, no-wash immunoassays using Alpha Streptavidin Donor and AlphaLISA Acceptor beads. In this technical note, we present the optimization of a signal decrease HDAC1 assay using as substrate a biotinylated Histone H3-derived peptide acetylated at lysine 27. In the absence of enzyme, the anti-H3K27ac Acceptor beads bind the acetylated residue on the peptide. Upon laser irradiation of the beads-target complexes at 680 nm, short-lived singlet oxygen molecules produced by the Donor beads can reach the Acceptor beads in proximity to generate maximal AlphaLISA signal at 615 nm (left panel). When the enzyme is added to the reaction, the peptide substrate is deacetylated and the anti-H3K27ac Acceptor beads do not recognize the biotinylated peptide anymore, leading to a signal decrease (right panel). This signal decrease is proportional to the deacetylation activity of the HDAC1 enzyme. Ac K B No Enzyme + Enzyme Ac K B K B + SA-Donor Bead + Ab-Acceptor Bead + SA-Donor Bead + Ab-Acceptor Bead No Emission Emission 615 nm Excitation 680 nm Excitation 680 nm Ac B K B Figure 1. Schematic representation of the AlphaLISA detection of a modified histone peptide. K Development of a HDAC1 Histone H3-Lysine 27 Deacetylase Assay Reagents needed for the assay: Anti-acetyl Histone H3 lysine 27 (H3K27ac) AlphaLISA Acceptor beads PerkinElmer # AL120 Histone H3 (21-44), H3K27ac peptide, biotinylated AnaSpec # 64846 Alpha Streptavidin Donor beads PerkinElmer # 6760002 AlphaLISA 5X Epigenetics Buffer 1 Kit PerkinElmer # AL008 HDAC1 (human), recombinant Cayman Chemical # 10009231 Trichostatin A Sigma # T8552 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 DTT, 0.01% Tween-20 and 0.01% BSA. 1.2 x 1006 Experiment 2: Enzyme Inhibition [HDAC1] (nM) 1,000,000 0 0.125 0.25 0.5 1 2 800,000 600,000 400,000 200,000 0 0 20 40 60 80 100 Time (min) 120 AlphaLISA Signal (counts) AlphaLISA Signal (counts) Experiment 1: Enzyme Titration and Time-Course Standard Protocol • Dilute HDAC1 enzyme, inhibitors and biotinylated Histone H3K27ac 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 RT – 5 μL of biotinylated Histone H3K27ac peptide substrate (2X) • Cover the plate with TopSeal-A film and incubate at room temperature (RT). • Prepare 1X Epigenetics Buffer 1 as recommended in the buffer technical data sheet. • Prepare a 5X Acceptor beads solution at 100 μg/mL in 1X Epigenetics Buffer 1 (final concentration of 20 μg/mL in 25 μL total assay volume). – 5 μL of Acceptor beads Addition of Acceptor beads prepared in 1X Epigenetics Buffer 1 stops the enzymatic reaction. • Cover with TopSeal-A film and incubate for 60 min at RT. • Prepare a 2.5X Streptavidin Donor beads solution at 50 μg/mL in 1X Epigenetics Buffer 1 (final concentration of 20 μg/mL in 25 μL total assay volume) in subdued light. – 10 μL of Streptavidin Donor beads • Cover with TopSeal-A film and incubate in subdued light for 30 min at RT. • Read signal in Alpha mode with the EnVision® or EnSpire® reader. 1,000,000 800,000 Trichostatin A IC50 = 3.2 nM 600,000 SAHA IC50 = 161 nM 400,000 200,000 0 -∞ -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log [Inhibitors] (M) Enzymatic progress curves were performed by incubating HDAC1 at concentrations ranging from 0.125 to 2 nM with 3 nM biotinylated Histone H3K27ac peptide substrate. Acceptor beads were added at the indicated times. Donor beads were added 60 min later and signal was read after 30 min. A 30 min reaction time using 1 nM enzyme was selected for all subsequent experiments. Serial dilutions of Trichostatin A ranging from 1 pM to 10 µM and serial dilutions of SAHA ranging from 10 pM to 100 µM were pre-incubated for 5 min with 1 nM of HDAC1. Enzymatic reactions were initiated by the addition of 3 nM biotinylated Histone H3K27ac peptide substrate. Enzymatic reactions contain 1% DMSO. AlphaLISA Signal (counts) Experiment 3: Z’-factor Determination 1.2 x 1006 1,000,000 100 nM Trichostatin A 800,000 600,000 Z' = 0.69 S/B = 2.9 No inhibitor 400,000 200,000 0 0 10 20 30 Well # 40 50 HDAC1 (1 nM) was pre-incubated with or without 100 nM Trichostatin A for 5 min. Enzymatic reactions were initiated by the addition of 3 nM biotinylated Histone H3K27ac peptide substrate. 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. 009614_01 Printed in USA Apr. 2011