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Assay Method Information

Assay Name:  Radioligand Binding Assay
Description:  HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Affinity data for this assay
 

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Last update November 1, 2007
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