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小木蟲論壇-學術科研互動平臺 » 生物醫(yī)藥區(qū) » 生物科學 » 蛋白/酶 » 關于酶活性檢測的問題
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[求助]
關于酶活性檢測的問題 已有1人參與
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關于酶的檢測的，文獻中說到的NADPH-generating system 是怎么參與到酶檢測的，起到的作用是什么？這段就沒怎么看懂，求能人指教 The incubation mixture, with a total volume of 200 μl, consisted of 100mM potassium phosphate buffer (pH 7.4), NADPH-generating system (1 mM NADP+, 10 mM glucose-6-phosphate, 1 unit/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), and human liver microsomes or CYPs. In all experiments, NCMN (20 mM dissolved in acetonitrile previously) was serially diluted to the required concentrations and the final concentration of acetonitrile did not exceed 1% (v/v) in the mixture. After preincubation at 37oC for 3 min, the reaction was initiated by adding NADPH-generating system and further incubated at 37oC in a shaking water bath. The reaction was terminated by the addition of ice-cold acetonitrile (100 μl). The mixture was kept on ice until it was centrifuged at 20,000×g for 20 min at 4oC. Aliquots of supernatants were stored at -20oC until analysis. Aliquots of supernatants were taken for further fluorescence analysis (Gain = 60 for substrate and Gain =100 for metabolite). Control incubations without NADPH-generating system or without substrate or without CYP enzyme sources were carried out to investigate whether the formation of metabolite(s) was enzyme (CYP)- and NADPH-dependent. All incubations throughout the study were carried out in duplicate with SD.values generally below 10%. |
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