TECHNICAL DATA SHEET AlphaScreen® Research Reagents Caution: For Laboratory Use. A research reagent for research purposes only cAMP AlphaScreen Assay Kit Product No.: 6760635D/M/R Lot No.: 2021216 Material Provided Format: 6760635D 6760635M 6760635R 1000 Assay Points 10000 Assay Points 50000 Assay Points Note: The number of assay points is based on a final bead concentration in a 25 µL/well reaction volume. Manufacturing Date: April 16, 2015 Kit Components: Component Anti-cAMP Acceptor Beads at 5 mg/mL in 1X PBS, 0.05% Proclin-300, pH 7.2 Streptavidin Donor Beads at 5 mg/mL in 1X PBS, 0.05% Proclin-300, pH 7.2 6760635D 1 x 100 µL 6760635M 1 x 1 mL 6760635R 1 x 5 mL (6760101B) (6760100B) (6760101) 1 x 100 µL 1 x 1 mL 1 x 5 mL (6760003B) (6760004) (6760004B) Biotinylated-cAMP Tracer Solid 1 x 10 nmol 1 x 10 nmol 1 x 65 nmol (6760301) (6760301) (6760301B) cAMP standard at 50 µM 1 x 1 mL 1 x 1 mL 1 x 1 mL (FP1283) (FP1283) (FP1283) 10x Buffer : AlphaLISA Immunoassay Buffer** 1 x 2 mL 1 x 10 mL 1 x 100 mL (6760040G) (6760040G) (6760040G) ** Extra buffer can be ordered separately (cat # AL000C: 10 mL, cat # AL000F: 100 mL). 1X PBS ph 7.4 is not included. It can be ordered from Gibco (cat# 10010-023) Product Information Antibody/Protein: The cAMP antibody is a rabbit monoclonal antibody highly specific for cyclic AMP. Its cross-reactivity with cGMP is < 0.005%. Stability: This product is stable for at least 6 months from the manufacturing date if used and stored under recommended conditions. Storage Conditions: Store undiluted at 4°C protected from light. Freeze-thaw is not recommended and can cause the beads to form aggregates. Recommended use: AlphaScreen® 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. TDS-6760625D/M/R-02 Page 1 of 3 ® For additional information on running AlphaScreen assays or on potential interfering compounds, please visit our website: www.perkinelmer.com/AlphaTech Quality Control ® AlphaScreen maximum signal, minimum signal and IC50 are determined by a cAMP competition assay performed ® on an EnVision HTS Alpha detection instrument. We certify that these results meet our requirements. TEST RESULTS Maximum signal Minimum signal LDL (nM) IC50 (nM) 297923 1277 1.461 5.944 Recommended Assay Conditions Note: This protocol provides a method to verify kit performance and is not representative of an assay. Sufficient biotinylated cAMP-Tracer and 10x buffer is provided to perform 3 competition curves in triplicate as described. ® 1x Buffer: Add 500 μL 10x buffer to 4.45 mL Milli-Q H2O (or equivalent). Acceptor Beads: Add 5 μL Anti-cAMP Acceptor beads to 495 μL 1x buffer. biotinylated cAMP-Tracer: Dissolve biotinylated cAMP-Tracer (10 nmol) in 1 mL 1X PBS (final 10 μM) Further dilute 10X in 1 X PBS, pH 7.4 to obtain a 1 M solution. *Solids may not be visible deposited along the walls of the tube* Donor beads: Add 6.25 μL Streptavidin Donor beads to 579.75 μL 1x buffer. cAMP standard: From the 50 µM cAMP standard, prepare a ½ log dilution series (5 µM to 50 pM) in 1x buffer. Include a buffer only control. Titration Protocol: To a white opaque 384-well Optiplate: 1- Add 10 μL of Anti-cAMP Acceptor beads. 2- Add 5 μL cAMP standard dilutions (from lowest to highest concentration). 3- Pre-mix the biotinylated cAMP-Tracer and Streptavidin Donor beads from previously diluted solutions by adding 39 µL of biotinylated cAMP-Tracer to the Streptavidin Donor beads dilution. Incubate the premix and the plate in the dark at room temperature for 30 minutes. 4- Add 10 μL of premix biotinylated cAMP-Tracer /Streptavidin Donor beads. ® Incubate in the dark at room temperature for 60 minutes and analyze on your AlphaScreen detection reader. TDS-6760625D/M/R-02 Page 2 of 3 Product Typical Data A lp h a S c r e e n S ig n a l (c o u n t s ) c A M P A lp h a S c r e e n 100000 80000 60000 40000 20000 0 -1 4 -1 2 -1 0 -8 -6 -4 [c A M P ] ( M ) Figure 1: Competition assay 384-well cAMP competition curve (25 μL final volume; Reader: Envision HTS Alpha). ® Note: AlphaScreen signal will vary depending on instrument detection protocol, incubation temperature and incubation time. Suggested Materials and Instrumentation Please visit our website 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. 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