TECHNICAL DATA SHEET AlphaLISA® Research Reagents Caution: For Laboratory Use. A research chemical for research purposes only TruHits Kit Product No.: AL900D AL900M Lot No.: 1643419 Product Formats Catalog # Assay points* AL900D 1 000 AL900M 10 000 * The number of assay points is based on an assay volume of 25 µL using a final bead concentration of 10 µg/mL in 384-well format. Manufacturing Date: June 21, 2012 Kit components: Component AL900D AL900M AlphaLISA BSA-biotin Acceptor beads stored in PBS, 0.05% Proclin-300, pH 7.2 1 x 50 µL at 5 mg/mL (AL900AD) 1 x 500 µL at 5 mg/mL (AL900AM) Streptavidin Alpha Donor beads stored in 25 mM Hepes, 100 mM NaCl, 0.05% Proclin-300, pH 7.4 1 x 50 µL at 5 mg/mL (6760002D) 1 x 500 µL at 5 mg/mL (6760002M) Product Information Description: The AlphaLISA Acceptor beads are coated with biotinylated bovine serum albumin (BSA). The Alpha Donor beads are coated with streptavidin. Application: This product is designed as a tool for AlphaLISA users to identify false positives in AlphaLISA screening assays. Storage: Store in the dark at 4ºC. Stability: This product is stable for at least 12 months from the manufacturing date when stored in its original packaging under recommended storage conditions. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. TDS-AL900-03 Page 1 of 4 Quality Control ® Lot-to-lot consistency is confirmed by a Quality Control one point assay read on an EnVision HTS Alpha instrument (see protocol below). We certify that the results meet our quality release criteria. Note: maximum counts will vary depending on assay conditions as well as between lots. This variation has no impact on assay quality. Maximum signal: Background: 875 926 counts 12 172 counts Description of the AlphaLISA TruHits Assay The AlphaLISA TruHits kit is designed as a tool for AlphaLISA users to identify false positives in AlphaLISA HTS assays early in the screening process. Some compounds can interfere with the detection part of the assay, thereby artificially reducing AlphaLISA signal. This kit includes AlphaLISA BSA-biotin Acceptor beads and Streptavidin Alpha Donor beads which interact together to generate an AlphaLISA signal. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm. The AlphaLISA TruHits kit allows the identification of inner filters, light scatterers (insoluble compounds), singlet oxygen quenchers and biotin mimetics interfering with the AlphaLISA signal, and thus facilitates the detection of false positives. However, TruHits kits do not allow systematic identification of all interfering compounds. Pan-assay interfering substances (PAINS) such as rhodanines will not cause a decrease in TruHits AlphaLISA signal. One Point Assay (Quality Control Protocol) This protocol provides a means to verify product performance. It is used as our Quality Control release test. The following reagents and materials are used: Item Suggested Source Catalog # Biotin Sigma B4501 (1X) Phosphate-Buffered Saline (PBS), pH 7.2 Invitrogen 20012 White OptiPlate™-384 PerkinElmer 6007290 TopSeal™-A Adhesive Sealing Film PerkinElmer 6050195 PerkinElmer - ® ® EnSpire or EnVision Multilabel Alpha Reader TDS-AL900-03 Page 2 of 4 Quality Control Protocol This protocol provides a method to verify kit performance and is not representative of an assay. The maximum and background signals represent an average of 6 replicates. Background signal is obtained in the presence of free biotin. Final concentration of AlphaLISA BSA-biotin Acceptor beads and Streptavidin Alpha Donor beads is 10 µg/mL, in the 25 µL final assay volume. Without Biotin With Biotin Maximum signal Background 1) Preparation of 1.7X Biotin (17 µM): Add 17 µL of 500 µM Biotin stock solution to 483 µL of 1X PBS, pH 7.2. 2) Preparation of 5X Streptavidin Alpha Donor beads (50 µg/mL): Keep the beads under subdued laboratory lighting. Add 5 µL of 5 mg/mL Alpha Donor beads to 495 µL of 1X PBS, pH 7.2. 3) Preparation of 5X AlphaLISA BSA-biotin Acceptor beads (50 µg/mL): Add 5 µL of 5 mg/mL AlphaLISA Acceptor beads to 495 µL of 1X PBS, pH 7.2. 4) In a microtube: Add 300 µL of 1X PBS, pH 7.2 Add 300 µL of 1.7X Biotin (10 µM final) Add 100 µL of 5X Alpha Donor beads (10 µg/mL final) Add 100 µL of 5X AlphaLISA Acceptor beads (10 µg/mL final) Incubate 30 min at 23ºC in the dark 5) In a white opaque OptiPlate-384 microplate: Distribute 25 µL in 6 replicates (-/+ biotin) Incubate 10 min at 23ºC in the dark Read using EnSpire or EnVision Multilabel Alpha Reader TDS-AL900-03 Page 3 of 4 Recommendations The volume indicated on each tube is guaranteed for single pipetting. Multiple pipetting of the reagents may reduce the theoretical amount left in the tube. To minimize loss when pipetting beads, it is preferable not to prewet the tip. Alpha Donor beads are light-sensitive. All Alpha assays using the Donor beads should be performed under subdued laboratory lighting (< 100 lux). Green filters (LEE 090 filters (preferred) or Roscolux filters #389 from Rosco) can be applied to light fixtures. Sodium azide should not be added to stock reagents. High concentrations of sodium azide (> 0.001 % final in the assay) might decrease the AlphaLISA signal. Centrifuge the tubes briefly before use to improve recovery of content (2,000 x g, 10-15 sec). Resuspend all reagents by vortexing before use. When reagents are added in the microplate, make sure the liquids are at the bottom of the well. Small volumes may be prone to evaporation. It is recommended to cover microplates with TopSeal-A Adhesive Sealing Film to reduce evaporation during incubation. Microplates are read with the TopSeal-A Film on the plate. Total signal varies with temperature and incubation time. For consistent results, identical incubation times and temperature should be used for all plates. The AlphaLISA signal is detected with an Alpha-enabled EnSpire or EnVision Multilabel Reader using the AlphaScreen standard settings (e.g. Total Measurement Time: 550 ms, Laser 680 nm, Excitation Time: 180 ms, Mirror: D640as, Emission Filter: M570w, Center Wavelength 570 nm, Bandwidth 100 nm, Transmittance 75%). Please visit our website for additional information on the AlphaLISA technology at www.perkinelmer.com/AlphaTech. This product is not for resale or distribution except by authorized distributors. LIMITED WARRANTY: PerkinElmer BioSignal Inc. warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and conform to specifications which accompany the product. PerkinElmer BioSignal Inc. makes no other warranty, express or implied with respect to the products, including any warranty of merchantability or fitness for any particular purpose. Notification of any breach of warranty must be made within 60 days of receipt unless otherwise provided in writing by PerkinElmer BioSignal Inc. No claim shall be honored if the customer fails to notify PerkinElmer BioSignal Inc. within the period specified. The sole and exclusive remedy of the customer for any liability of PerkinElmer BioSignal Inc. of any kind including liability based upon warranty (express or implied whether contained herein or elsewhere), strict liability contract or otherwise is limited to the replacement of the goods or the refunds of the invoice price of goods. PerkinElmer BioSignal Inc. shall not in any case be liable for special, incidental or consequential damages of any kind. 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. 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