| 查看: 2147 | 回复: 0 | ||
laoyongmin新虫 (初入文坛)
|
[求助]
怎么测表观Km常数？
|
|
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 |
回复此楼» 猜你喜欢
266求调剂
已经有4人回复
-
324求调剂
已经有9人回复
-
招收生物学/细胞生物学调剂
已经有4人回复
-
一志愿武汉理工，总分321，英一数二，求老师收留。
已经有5人回复
-
284求调剂
已经有12人回复
-
348求调剂
已经有3人回复
-
329求调剂
已经有7人回复
-
求调剂，一志愿 南京航空航天大学大学 ，080500材料科学与工程学硕
已经有4人回复
-
考研调剂
已经有9人回复
-
304材料求调剂
已经有4人回复
