Affinity small molecules for the EPO receptor
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[0139] The initial competitive ERP screening assay was designed as a solid plate binding assay. A detailed protocol of the assay is described below in the Protocolfor ERP Screen section.
[0140] Briefly, in the competitive ERP screening assay, the extracellular portion of EPO-R was purchased from R&D (Minneapolis, Minn.); biotinylated and regular peptide were synthesized by American Peptide Company; receptor and biotinylated peptide (bio-ERP) were incubated at room temperature; the solution was contacated with neutravidin (streptavidin) coated plate to bind any complex between receptor and peptide; the complex that was bound to the plate is detected by antibody specific for EPO-R and detection is performed with HRP-conjugated secondary antibody; the presence of the complex between receptor and peptide is detected by an light absorbance at 490 nm; and lack of or low signal indicates that the biotinylated peptide has been competed out, indicating an affinity of the test compound for the...
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