T E C H N I C A L N O T E U-TRF #49 LANCE Ultra Tau-Ser400 O-GlcNAc hydrolase (OGA) Assay Marie-Élaine Caruso Mireille Caron Nancy Gauthier Anja Rodenbrock Philippe Bourgeois Liliana Pedro Lucille Beaudet Roberto Rodriguez-Suarez LANCE® Ultra TR-FRET Technology PerkinElmer, Inc. Montreal, QC Canada H3J 1R4 This LANCE Ultra immunodetection assay measures the hydrolysis of an O-GlcNAc moiety from a biotinylated Tau-Ser400-O-GlcNAc peptide. LANCE Ultra Europium-anti-O-linked-GlcNAc Antibody: • TRF0413-D: 10 µg, 1,562 assay points* • TRF0413-M: 100 µg, 15,625 assay points* *6.7 fmol/assay point Peptide Sequence: KWKHGAEIVYKSPVV-S(O-GlcNAc)-GDTSPRHLSNVK-K(biotin)-NH2 GlcNac B S/T No Enzyme + O-GlcNAc Hydrolase GlcNac B S/T B S/T + Eu-Ab + ULight-SA + Eu-Ab + ULight-SA FRET Eu Excitation 320 or 340 nm No FRET Eu GlcNac ULight B S/T ULight B S/T Em 665 nm Figure 1. Schematic representation of the LANCE Ultra detection of an O-GlcNAcylated peptide (B: biotin group; S/T: serine or threonine residue). Excitation 320 or 340 nm 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 OGA signal-decrease assay using as substrate a biotinylated Tau-derived peptide O-GlcNAcylated at Ser400. In this assay, detection of the non-hydrolyzed O-GlcNAcylated substrate was performed by the addition of 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 signal decrease is proportional to the activity of the OGA enzyme. Development of a LANCE Ultra Tau-Ser400 O-GlcNAc hydrolase (OGA) Assay Experiment 1: Enzymatic Titration and Time-Course Reagents needed for this assay: Europium-anti-O-linked-GlcNAc Antibody PerkinElmer # TRF0413 LANCE Ultra ULight-Streptavidin PerkinElmer # TRF0102 Tau-Ser400-O-GlcNAc (388-411), biotinylated AnaSpec # 65409 LANCE Detection Buffer, 10X PerkinElmer # CR97-100 O-GlcNAcase (S. pyogenes), recombinant (OGA) Prozomix # PRO-E0255 White opaque OptiPlate™-384 PerkinElmer # 6007290 TopSeal -A film PerkinElmer # 6050195 O-(2-Acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc) Carbosynth # EA06838 N6-Methyladenine Carbosynth # FM10151 ™ Assay buffer: 20 mM MES, pH 6, 0.05% BSA and 0.01% Tween Enzymatic progress curves were performed by incubating OGA at concentrations ranging from 0.125 to 3 nM with 250 nM biotinylated Tau-Ser400-O-GlcNAc peptide substrate. 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 1 nM enzyme was selected for all subsequent experiments. Experiment 2: Enzyme Inhibition Standard Protocol • Dilute OGA, inhibitors and biotinylated biotinylated Tau-Ser400-O-GlcNAc peptide substrate in Assay Buffer just before use. • Add to the wells of a white OptiPlate-384: – 2.5 µL of inhibitor (4X) or Assay Buffer – 2.5 µL of enzyme (4X) – Incubate for 5 min at room temperature (RT). – 5.0 µL biotinylated Tau-Ser400-O-GlcNAc peptide (2X) • Cover the plate with TopSeal-A film and incubate at RT. • Prepare Detection Mix by diluting the Eu-Ab to 0.67 nM and ULight-Streptavidin to 100 nM in 1X LANCE Detection Buffer (final concentrations of 0.33 nM and 50 nM in 20 µL total assay volume). • Add 10 µL of Detection Mix. Addition of the Detection mix will stop the enzymatic reactions. Serial dilutions of PUGNAc and N6-Methyladenine ranging from 100 pM to 10 µM and 10 nM to 1 mM, respectively, were pre-incubated for 5 min with 1 nM OGA. Enzymatic reactions were initiated by the addition of 250 nM biotinylated Tau-Ser400-O-GlcNAc peptide substrate. Enzymatic reactions contain 1% DMSO. Experiment 3: Z’-factor Determination • Cover with TopSeal-A film and incubate 60 min at RT. • Remove the TopSeal-A film and read signal with the EnVision® Multilabel Plate Reader in TR-FRET mode (excitation at 320 or 340 nm and emission at 665 nm). OGA (1 nM) was pre-incubated with or without 1 µM PUGNAc or 250 µM N6-Methyladenine for 5 min. Enzymatic reactions were initiated by the addition of 250 nM biotinylated Tau-Ser400-O-GlcNAc 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 ©2007-2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 010816_01 Printed in USA Dec. 2012