TECHNICAL DATA SHEET Membrane Target Systems™ Caution: For Laboratory Use. A research reagent for research purposes only human Adenosine A2A Receptor Product No.: RBHA2AM400UA Lot No.: 1584001 Material Provided Membranes: 1 x 400 units / 400 µL frozen aliquot Product Information Cellular Background: HEK293 GenBank Accession Number: NM_000675 Unit Size: 9 µg protein / unit Storage Buffer: 50 mM Tris-HCL (pH 7.4), 0.5mM EDTA, 10mM MgCl2, 10% sucrose. Storage Conditions: Store at -80°C. Freeze-thaw is not recommended as it can affect product performance and homogeneity. In order to minimize negative impact of freeze-thawing, flash freeze in liquid o nitrogen for 30 seconds prior to transferring to -80 C. Stability: This product is stable for at least 3 years from reception if used and stored under recommended conditions. Quality Control Bmax and Kd are determined using radioactive saturation binding assays (Figure 1). Protein (1) concentration is determined using the BCA method . Ratio-to-Reference (RTR) is determined by dividing the maximal signal of the current lot (Bmax in fmoles) by the maximal signal of a pre-defined reference tested in parallel. RTR is an indicator of lot-to-lot consistency. *We certify that these results meet our quality release criteria. Ratio-to-Reference (RTR): 1.21 Expression Level (BMAX): 14 pmol/mg membrane protein. 3 KD for [ H]-CGS 21680: 29 nM Protein Concentration: 9 µg/µL (1) Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85. TDS-RBHA2AM400UA-03 Page 1 of 4 Recommended Assay Conditions Assay Buffer: 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 µg/mL Adenosine Deaminase Wash Buffer: 50 mM Tris-HCl pH 7.4, 154 mM NaCl Binding Protocol: Binding assays are performed in 550 µL total volume according to the following conditions: 1 - Membrane dilution: 0.05 mL of membranes + 24.95 mL assay buffer (1:500 dilution) 25 µL of incubation buffer or 5′-(N-Ethylcarboxamido)adenosine (Sigma E2387) 50 µM final for non specific binding (Saturation binding assay) 2 - Incubation: For competition binding assay: 25 µL of reference compounds at decreasing concentrations (see figure 2) 25 µL of radioligand at the appropriate concentration (see graph below) 500 µL of diluted membranes 3 - Incubation time: 90 minutes at 27 °C 4 - Filtration: aspirate and wash 9 x 500 µL with ice cold wash buffer over GF/C filter (presoaked in 0.5 % PEI). Lot Specific Data 9000 Total Non specific Specific 8000 Bound (cpm) 7000 6000 5000 4000 3000 2000 1000 0 0 25 50 75 100 125 150 [3H]-CGS 21680 (nM) Figure 1: Saturation binding assay curve (filtration) 96-well saturation binding assay curve (9 µg 3 membranes/well, TopCount®) using [ H]-CGS 21680 (PerkinElmer NET1021 Lot No.: 619326) TDS-RBHA2AM400UA-03 Page 2 of 4 Typical Product Data Bound (cpm) 4000 CGS-21680 3000 (-)-N6-(2-Phenylisopropyl)adenosine 2000 (+)-N6-(2-Phenylisopropyl)adenosine 1000 0 - -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 [Compound] (M) Figure 2: Competition binding assay curve (filtration) 96-well competition binding assay curve (9 µg membranes/well, TopCount®). Recommended radioligand concentration = 50 nM. *Even though two sites can be observed occasionally with some ligands, the data presented is derived from single site fitting. Reference Compounds CGS-21680 6 658 6 12073 (−)-N -(2-Phenylisopropyl)adenosine (+)-N -(2-Phenylisopropyl)adenosine TDS-RBHA2AM400UA-03 Page 3 of 4 Ki (nM) 47 Suggested Materials and Instrumentation Please visit our website www.perkinelmer.com/GPCR 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. 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