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Primary and Secondary Binding Experiments. Radioligand binding assays using cloned GPCRs, ion channels, and transporters were performed using membranes from transiently transfected or stable cell lines as previously detailed(1, 46) through the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. Detailed protocols (including cell handling, buffer composition, assay conditions, etc.) for all assays are available online.(https://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf) Initial screening assays were performed using a 10-μM (final concentration)test compound, and the percent inhibition of specific binding by the test compound was determined. When the test compound inhibited >50% of radioligand specific binding, Ki determinations were performed by measuring the inhibition of radioligand binding by various concentrations of test drug (11 concentrations spanning six orders of magnitude). Radioligand binding isotherms were regressed using the One Site Competition Binding function built into Prism 4.0 (GraphPad) to estimate compound IC50 values. Affinity constants (Ki values) were calculated from IC50 values using the Cheng–Prusoff approximation. Representative Procedures. |
木蟲 (正式寫手)
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結(jié)合首要和次要實驗方案,放射性配體結(jié)合使用克隆GPCRs檢測法、離子通道和轉(zhuǎn)運法用于短暫轉(zhuǎn)染或穩(wěn)定細胞系的細胞膜,詳細參照(1,46)通過美國國家精神衛(wèi)生研究院關(guān)于精神刺激藥物篩選程序的資源。所有試驗的詳細協(xié)議(包括細胞處理,緩沖組成,測定條件等)可參照.(https://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf) 首要篩選試驗使用一個10-μM得測試化合物(末濃度),抑制百分比的特殊結(jié)合性通過測試化合物來決定,當測試化合物的抑制百分比>50%放射性配體時,通過各種濃度的測試藥物(11濃度采用六個數(shù)量級)測量放射性配體的抑制百分比來決定Ki,放射性配體的等溫線反復使用一個地址競爭結(jié)合作用建立棱鏡4.0(GraphPad)來估算化合物IC50 的值,親和常數(shù)(Ki值)可以通過Cheng–Prusoff 近似法得到的IC50值計算出來。 有代表性的程序。 |
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