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laoyongmin新虫 (初入文坛)
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[求助]
怎么测表观Km常数？
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目前在纯化His-tagged蛋白，马上做酶性质分析，想测表观Km（apparent kinetic constants），参考文献（和我一样的酶）：“For determination of apparent kinetic constants, eight concentrations each of the substrates DMADP (0–100 uM), GDP (0–50 uM), FDP (0–50 uM) or GGDP (0–50 uM) were used in the presence of a fixed IDP concentration (100 uM). The assay mixtures contained 25 mM MOPSO (pH 7.2), 10 mM MgCl2 and 10% v/v glycerol, plus IDP and DMADP, GDP, FDP or GGDP and 1 ug purified AtIDS9 per assay in a 200 uL volume. The reaction mixture was incubated in glass vials at 20°C for times ranging from 0–2 min in 10 sec intervals with shaking using an Eppendorf Thermomixer Comfort (www.eppendorf.com), and the reaction was stopped by immediately freezing the sample in liquid nitrogen. To determine the GFDP concentration, samples were analyzed via UPLC/MS using a calibration curve”，这里写的8个浓度和反应时间（0-2 min）该如何设置？文献的结果如图所示，有些只有7和点，不是有8个浓度吗？图中的1/V和1/S是怎样算出来的？ Figure S2: Lineweaver-Burk plots for calculation of apparent in vitro Km values of AtIDS9 with different substrates. The double reciprocal velocity (1/v) and substrate concentrations (1/[S]) are plotted to determine the apparent Km values. A. DMADP (0-100 µM) + 100 µM IDP used as substrate B. GDP (0-50 µM) + 100 µM IDP used as substrate C. FDP (0-50 µM) + 100 µM IDP used as substrate. D. GGDP (0-15 µM) + 100 µM IDP used as substrate. E. IDP (0-40 µM) + 50 µM GGDP used as substrate.  11.jpg |
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