T E C H N I C A L N O T E AlphaLISA #10 AlphaLISA SIRT1 p53 Lysine 382 Deacetylase Assay Authors Mathieu Arcand Julie Blouin Mireille Caron Claire Normand Anne Labonté Lucille Beaudet Jaime Padrós AlphaLISA® PerkinElmer, Inc. Montreal, QC Canada, H3J 1R4 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 SIRT1 assay using as substrate a biotinylated p53-derived peptide acetylated at lysine 382. In the absence of enzyme or cofactor, the anti-p53K382ac 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 enzyme and cofactor are added to the reaction, the peptide substrate is deacetylated and the anti-p53K382ac 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 SIRT1 enzyme. This AlphaLISA immunodetection assay measures the deacetylation of a biotinylated p53 (368-393) peptide acetylated at lysine 382. Anti-acetyl-p53 Lysine 382 (p53K382ac) AlphaLISA® Acceptor Beads • AL124C: 250 µg, 500 assay points* • AL124M: 5 mg, 10,000 assay points* • AL124R: 25 mg, 50,000 assay points* *0.5 µg/assay point Peptidic Substrate Sequence: (Biotin)K-GG-HLKSKKGQSTSRHKK(ac)LMFKTEGPDSD-NH2 Ac K B No Enzyme + Enzyme NAD+ 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 p53-derived peptide. K Development of a SIRT1 p53 Lysine 382 Deacetylase Assay: Reagents needed for the assay: Anti-acetyl-p53 Lysine 382 AlphaLISA Acceptor beads PerkinElmer # AL124 p53 (368-393) acetyl-lysine 382 peptide (p53K382ac), biotinylated AnaSpec # 64869 Alpha Streptavidin Donor beads PerkinElmer # 6760002 AlphaLISA 5X Epigenetics Buffer 1 Kit PerkinElmer # AL008 Sirtuin 1 (human), recombinant BPS BioScience # 50012 EX-527 Tocris # 2780 Suramin Calbiochem # 574625 SIRT1 inhibitor III Calbiochem # 566322 Nicotinamide Sigma # N3376 ß-Nicotinamide adenine dinucleotide hydrate (NAD+) Sigma # N1636 White opaque OptiPlate™-384 PerkinElmer # 6007299 TopSeal™-A films PerkinElmer # 6005185 Assay Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 0.01% Tween-20 and 0.01% BSA. 250,000 Experiment 3: Enzyme Inhibition [SIRT1] (nM) 200,000 0 0.25 0.5 1 2 5 150,000 100,000 50,000 0 0 20 40 60 80 Time (min) 100 120 AlphaLISA Signal (counts) AlphaLISA Signal (counts) Experiment 1: Enzyme Titration and Time-Course Standard Protocol • Dilute SIRT1 enzyme, inhibitors, biotinylated p53K382ac peptide substrate and NAD+ 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 – 2.5 μL of biotinylated p53K382ac peptide substrate (4X) – 2.5 µL of NAD+ (4X) • 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 Stop Solution Mix containing 250 µM of EX-527 and 100 μg/mL of Acceptor Beads in 1X Epigenetics Buffer 1 (final concentration of 50 µM EX-527 and 20 μg/mL Acceptor Beads in 25 μL total assay volume). – 5 μL of Stop Solution Mix • 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. 200,000 EX-527 IC50 = 1.09 µM 150,000 SIRT1 Inhibitor III IC50 = 0.81 µM 100,000 Suramin IC50 = 0.13 µM 50,000 0 Nicotinamide IC50 = 91 µM -∞ -9 -8 -7 -6 -5 Log [Inhibitor] (M) -4 -3 -2 Experiment 2: NAD+ Titration Experiment 4: Z’-factor Determination 250,000 200,000 150,000 100,000 Km app = 167 µM 50,000 0 -∞ -7 -6 -5 -4 Log [NAD+] (M) -3 -2 Serial dilutions of NAD+ ranging from 300 nM to 12.5 mM were added to 0.5 nM SIRT1 and 3 nM biotinylated p53K382ac peptide substrate. Enzymatic reactions were incubated for 30 min. A 200 µM NAD+ concentration was selected for subsequent experiments. AlphaLISA Signal (counts) Serial dilutions of inhibitors ranging from 1 nM to 100 µM (EX-527), 3 nM to 100 µM (SIRT1 Inhibitor III), 10 nM to 10 µM (suramin) and 300 nM to 3 mM (nicotinamide) were pre-incubated for 5 min with 0.5 nM of SIRT1. Enzymatic reactions were initiated by the addition of 3 nM biotinylated p53K382ac peptide substrate and 200 µM NAD+. Enzymatic reactions contained 1% DMSO and proceeded for 60 min. AlphaLISA Signal (counts) Enzymatic progress curves were performed by incubating SIRT1 at concentrations ranging from 0.25 to 5 nM with 3 nM biotinylated p53K382ac peptide substrate and 2 mM NAD+. A mix of Acceptor beads and EX-527 was added to stop the reaction at the indicated times. Streptavidin Donor beads were added 60 min later and signal was read after 30 min. A 0.5 nM enzyme was selected for all subsequent experiments. 200,000 30 µM EX-527 Z' = 0.55 SB = 2.6 150,000 100,000 50,000 0 No inhibitor 0 10 20 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. Printed in USA Apr. 2011 40 50 SIRT1 (0.5 nM) was pre-incubated with or without 30 µM EX-527 for 5 min. Enzymatic reactions were initiated by the addition of 3 nM biotinylated p53K382ac peptide substrate and 200 µM NAD+. Enzymatic reactions contained 1% DMSO and proceeded for 60 min. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com 009615_01 30 Well #