TECHNICAL DATA SHEET Membrane Target Systems™ Caution: For Laboratory Use. A research reagent for research purposes only human Muscarinic M3 Receptor Product No.: RBHM3M400UA Lot No.: XXX-XXX-X Material Provided Membranes: 1 x 400 units / 1000 µL frozen aliquot Product Information Cellular Background: CHO-K1 GenBank Accession Number: NM_000740 Unit Size: 15 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): N/A Expression Level (BMAX): 11.98 pmol/mg membrane protein. 3 KD for [ H]-Scopolamine (N-methyl) : 0.15 nM Protein Concentration: (1) 6 µg/µL Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85. TDS-RBHM3M400UA-04 Page 1 of 4 Recommended Assay Conditions Assay Buffer: PBS pH 7.4 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: 0.125 mL of membranes + 24.875 mL assay buffer (1:200 dilution) 2 - Incubation: 25 µL of incubation buffer or Atropine (Sigma A025) 5 µM final for non specific binding (Saturation binding assay) 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: 120 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 30000 T o ta l B o u n d (c p m ) N o n s p e c ific S p e c ific 20000 10000 0 0 1 2 3 4 5 6 7 8 9 10 [ 3 H ] - S c o p o la m in e ( N - m e t h y l) ( n M ) Figure 1: Saturation binding assay curve (filtration) 96-well saturation binding assay curve (15 µg 3 membranes/well, TopCount®) using [ H]-Scopolamine (N-methyl) (PerkinElmer NET636 Lot No.: 1885805) TDS-RBHM3M400UA-04 Page 2 of 4 Typical Product Data Bound (cpm) 5000 4000 p-Fluorohexahydro-sila-difenidol 3000 Pirenzepine 2000 Methoctramine 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 (15 µg membranes/well, TopCount®). Recommended radioligand concentration = 0.6 nM. *Even though two sites can be observed occasionally with some ligands, the data presented is derived from single site fitting. Reference Compounds p-Fluorohexahydro-sila-difenidol TDS-RBHM3M400UA-04 Page 3 of 4 Ki (nM) 87 Pirenzepine 637 Methoctramine 1997 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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