TECHNICAL DATA SHEET Membrane Target Systems™ Caution: For Laboratory Use. A research reagent for research purposes only human Adenosine A2B Receptor Product No.: ES-013-M400UA Lot No.: 1648739 Material Provided Membranes: 1 x 400 units / 1000 µL frozen aliquot Product Information Cellular Background: HEK293 GenBank Accession Number: M97759 Unit Size: 20 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): 1.3 Expression Level (BMAX): 0.86 pmol/mg membrane protein. 3 KD for [ H]-Cyclopentyl-1,3-dipropylxanthine : 36.5 nM Protein Concentration: (1) 8 µg/µL Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85. TDS-ES-013-M400UA-04 Page 1 of 4 Recommended Assay Conditions Assay Buffer: 50 mM Hepes pH 7.4, 5 mM MgCl2, 1 mM EDTA Wash Buffer: 5 mM Hepes pH 6.5, 5 mM MgCl2, 1 mM EDTA, 0.25% BSA Binding Protocol: Binding assays are performed in 550 µL total volume according to the following conditions: 1 - Membrane dilution: 0.125 mL of membranes + 24.875 mL assay buffer (1:200 dilution) 25 µL of incubation buffer or 5′-(N-Ethylcarboxamido)adenosine (Sigma E2387) 100 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: 60 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 % BSA). Lot Specific Data Bound (cpm) 3000 Total Non specific Specific 2000 1000 0 0 10 20 30 40 50 60 [3H]-Cyclopentyl-1,3-dipropylxanthine (nM) Figure 1: Saturation binding assay curve (filtration) 96-well saturation binding assay curve (20 µg 3 membranes/well, TopCount®) using [ H]-Cyclopentyl1,3-dipropylxanthine (PerkinElmer NET974 Lot No.: 1637680) TDS-ES-013-M400UA-04 Page 2 of 4 Typical Product Data Bound (cpm) 1000 750 5'-(N-Ethylcarboxamido)adenosine 500 250 0 - -11 -10 -9 -8 -7 -6 -5 -4 -3 [Compound] (M) Figure 2: Competition binding assay curve (filtration) 96-well competition binding assay curve (20 µg membranes/well, TopCount®). Recommended radioligand concentration = 5 nM. *Even though two sites can be observed occasionally with some ligands, the data presented is derived from single site fitting. TDS-ES-013-M400UA-04 Page 3 of 4 Reference Compounds Ki (nM) 5′-(N-Ethylcarboxamido)adenosine 880 Suggested Materials and Instrumentation Please visit our website www.perkinelmer.com/GPCR This product is not for resale or distribution except by authorized distributors. When applicable, the CMV promoter is covered by U.S. Patents 5,168,062 and 5,385,839, licensed to PerkinElmer by the University of Iowa research Foundation 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. 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