TECHNICAL DATA SHEET AlphaScreen® Research Reagents Caution: For Laboratory Use. A research reagent for research purposes only Phosphotyrosine (PY20) Detection Kit Product No.: 6760601C/M/R Lot No.: 1823834 Material Provided Format: 6760601C 6760601M 6760601R 500 Assay Points 10000 Assay Points 50000 Assay Points Note: The number of assay points is based on a final bead concentration of 20 µg/mL in a 25 µL/well reaction volume. Manufacturing Date: October 29, 2013 Kit Components: Component Anti-phosphotyrosine (PY20) Acceptor Beads at 5 mg/mL in 25 mM Hepes, 100 mM NaCl, 0.05% Proclin-300, pH 7.4 Streptavidin Donor Beads at 5 mg/mL in 25 mM Hepes, 100 mM NaCl, 0.05% Proclin-300, pH 7.4 Biotinylated-LCK-P at 5 µM in 25mM Hepes, 0.05% Proclin-300, pH 7.4 10x Buffer : 250 mM Hepes, 1 M NaCl, 0.05% Proclin-300, pH 7.4 6760601C 6760601M 6760601R 1 x 50 µL 1 x 1 mL 1 x 5 mL (6760102) (6760103) (6760103B) 1 x 50 µL 1 x 1 mL 1 x 5 mL (6760001) (6760002) (6760002B) 1 x 50 µL 1 x 50 µL 1 x 50 µL (6760252) (6760252) (6760252) 1 x 1.5 mL 1 x 1.5 mL 1 x 1.5 mL (6760020G) (6760020G) (6760020G) Product Information Antibody/Protein: The PY20 antibody is a mouse monoclonal antibody (IgG2b) that binds to tyrosine phosphorylated proteins in the native or denatured states. Stability: This product is stable for at least 12 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-6760601C/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 EC50 are determined using a biotinylated-LCK-P titration assay performed on an EnVision ® HTS Alpha detection instrument. We certify that these results meet our requirements. TEST RESULTS Maximum signal Minimum signal EC50 784116 660 5.23 Recommended Assay Conditions Note: This protocol provides a method to verify kit performance and is not representative of an assay. Sufficient biotinylated-probe and 10x buffer is provided to perform 3 titration curves in triplicate as described. ® 1x Buffer: Add 500 μL 10x buffer to 4.5 mL Milli-Q H2O (or equivalent). Add 5 mg BSA (0.1% final concentration) and adjust pH to 7.4. Acceptor Beads: Add 5 μL Anti-phosphotyrosine (PY20) Acceptor beads to 245 μL 1x buffer. Donor beads: Add 5 μL Streptavidin Donor beads to 245 μL 1x buffer. Biotinylated-probe: From the 5 µM biotin-LCK-P, prepare a ½ log dilution series (0.5 µM to 50 pM) in 1x buffer. Include a buffer only control. Titration Protocol: To a white opaque 384-well Optiplate: 1- Add 5 μL biotinylated-LCK-P dilutions (from lowest to highest concentration). 2- Add 10 μL of 1x buffer 3- Pre-mix (1:1) the Anti-phosphotyrosine (PY20) Acceptor beads and Streptavidin Donor beads from previously diluted solutions by adding 200 µL Anti-phosphotyrosine (PY20) Acceptor beads to 200 µL Streptavidin Donor beads. 4- Add 10 μL of pre-mix (1:1) of Anti-phosphotyrosine (PY20) Acceptor beads and Streptavidin Donor beads. Incubate in the dark at room temperature for 60 minutes and analyze on your AlphaScreen detection reader. TDS-6760601C/M/R-02 Page 2 of 3 AlphaScreen signal (counts) Product Typical Data 750000 600000 450000 300000 150000 0 - -11 -10 -9 -8 -7 -6 log [b-LCK-P] (M) Figure 1: Biotinylated-probe titration assay 384-well biotinylated-LCK-P titration 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 includi ng 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 ©2009, PerkinElm er, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.