TECHNICAL DATA SHEET AlphaScreen® Research Reagents Caution: For Laboratory Use. A research reagent for research purposes only Conjugation Kit Product No.: 6760000K Lot No.: 1972377 Material Provided Format: 6760000K 2 x 2 mg* * Note: 1mg translates into 2,000 assay points is based on a final bead concentration of 20 µg/mL in a 25 µL/well reaction volume. Manufacturing Date: December 1, 2014 Kit Components: Component Unconjugated Acceptor Beads at 20 mg/mL in 0.1M MES, pH 6.0 Streptavidin Donor Beads at 5 mg/mL in 25mM Hepes, 0.1M NaCl, 0.05% proclin, pH 7.4 6760000K 2 x 50 µL (6762001) 2 x 200 µL (6760002S) Product Information 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. ® For additional information on running AlphaScreen assays or on potential interfering compounds, please visit our website: www.perkinelmer.com/AlphaTech Conjugation Protocol Note: The following procedure is recommended only as a general guideline for bead conjugation to IgG molecules. It is highly recommended that conditions are further optimized as required for the application for which the beads are intended. TDS-6760000k-05 Page 1 of 2 Materials: Aldehyde beads: (Provided) Sodium cyanoborohydride solution: Dissolve 25 mg of sodium cyanoborohydride (Sigma #15,615-9 or equivalent) ® in 1 mL Milli-Q H2O or equivalent. Note: Prepare fresh. MES buffer (0.1 M pH 6.0): Dissolve 2.13g MES (2-[N-Morpholino]ethanesulfonic Acid) (Sigma # M-5287 or ® equivalent) in 80 mL Milli-Q H2O or equivalent. Adjust the pH to 6.0 with 10 N NaOH and make up to 100 mL total volume. Filter through a 0.2 μm filter. 1% Tween-20: Dilute 10% Tween-20 (Pierce #28320 or equivalent) 1/10 in MES buffer. CMO solution (0.3 M pH 5.0): Dissolve 65 mg CMO (carboxymethoxylamine hemihydrochloride) (Sigma #C1,340-8 ® or equivalent) in 1 mL Milli-Q H2O or equivalent. Adjust the pH to 5.0 with 10 N NaOH. Note: Prepare fresh. TRIS buffer (0.1 M pH 8.0): Dissolve 1.21g Tris (ICN #819638 or equivalent) in 80mL deionised H2O. Adjust the pH to 8.0 with HCl and make up to 100mL total volume. Filter through a 0.2μm filter. Conjugation: 1. To each tube (1 mg beads) provided add: 12.5 μL 1% Tween-20 0.05 mg antibody (This will provide a 20:1 bead:antibody wt:wt ratio. As antibodies are available in different concentrations depending on the supplier, 50 μL of a 1 mg/mL solution is recommended) 10μL Sodium cyanoborohydride solution o 2. Make up to a total volume of 200 μL with 0.1 M MES pH 6.0. Incubate in the dark for 48 hours at 37 C. Blocking: o Add 10 μL CMO solution. Incubate in the dark at 37 C for 1 hour. Purification: 1. Add 190 μL 0.1 M Tris buffer pH 8.0. o 2. Centrifuge at 13,000 g for 30 min at 4 C. 3. Remove the supernatant with a micropipette. 4. Add 1 mL 0.1 M Tris buffer pH 8.0 to pellet. 5. Vortex and sonicate*. o 6. Centrifuge at 13,000 g for 30 min at 4 C. 7. Repeat steps 3 through 6. 8. Add 200 μL of an appropriate buffer** (5 mg/mL final concentration). 9. Vortex and sonicate*. o 10. Store in the dark at 4 C *Sonicate on ice at 10 pulses of 1 second (0.5 second ON/0.5 second OFF) using a Misonix XL sonicator (Model CL4) and HS419 microtip probe. ** Either PBS or 25 mM Hepes, 100 mM NaCl, pH 7.4 is recommended. 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 ©2009, PerkinElmer, Inc. All rights reserved. PerkinElmer ® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.