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[求助]
请教微管蛋白实验
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tubulin protein assay 这个实验怎么做啊。看的一个AM的文章做的检测PTX的实验，有些不明。求教。 Relative activities of PTX were estimated by calculating the maximum rate of tubulin protein polymerization relative to the standard samples (Figure S5). Stabilities of the free and encapsulated PTX inside different types of NCs were evaluated by measuring the variations of their activities using the tubulin assay after storage at 25 °C and 37 °C for 1–14 d in 96-well microtiter plates according to previously established protocols. Briefly, at defined time intervals, NCs were separated using centrifugation and redispersed in water at pH 4.5 to extract PTX from NCs, and then PTX was separated using HPLC to perform a tubulin protein assay. Then, select concentrations of PTX were reacted with tubulin protein solution (general tubulin buffer, tubulin glycerol buffer, 1 mM GTP), and the reaction was followed by measuring the increase in apparent absorption at 350 nm over a 1 h period at 37 °C using an ELISA plate reader (Safire II, Tecan Sales Switzerland AG, Mannedorf, CH). As shown in Figure S5, drug degradation is generally considered as a first order process and can be described by the following equation: ln(a/a0) = kt, where a is the activity of drug at time t, a0 is the initial drug activity, k is the degradation rate constant. At half-life of drug (t1/2), we have a=a0/2 => t1/2= 0.693/k => k=0.693/t1/2. The results of PTX half-life (t1/2) and degradation rate constant (k) are shown in Figure S6 and Figure 3(a), respectively. |
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