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[求助]
挥发油抑制酪氨酸酶的溶解性问题
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| 请问有没有人做挥发油的酪氨酸酶抑制试验，我的样品室挥发油，溶解在DMSO或乙醇中，但是当PBS缓冲液加在样品以后，DMSO或乙醇浓度降低，不足以是挥发油溶解，导致挥发油析出，呈现为小油滴，使得反应体系浑浊，同时也不可测得挥发油真实的抑制率，求哪位大侠遇到过这种问题，解释这一现象，并解决 |
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Set up progressive dilution of the essential oil (EO) in Ethanol 60% (start from an original dilution od 1:100 corresponding to about 0.0087 g/mL of oil and then progressively dilute 2-1.5 fold in EtOH 60%, at each dilution vortex extensively. Vortex well the dilution just before adding it to 200 ul of final reaction: add 10 uL of the dilution, but you can even add 5 uL in 200 uL to improve solubility (final EtOH 1,5%). When the enzyme is added to start reaction, mix very well again by pipetting in the single wells and by programming a shaking of the plate in the plate-reader instrument, if possible. Some cloudiness may be neglected. Indeed, as we monitored the delta absorbance per time, the initial absolute absorbance was almost irrelevant: what you need is 1) the reaction takes place; 2)the difference value between Abs at time zero and Abs at the end. I have also experienced some problem in solubility. Actually final concentration above 430 ug/mL could not be achieved and, in some replicas, at these high concentrations (about 400ug/mL) solution could look cloudy: just mix very well by pipetting and use the correct incubation temperature; because your oil might be slightly different in composition from what I used, and this could affect solubility, too, I suggest you could use dilutions of 350-200 ug/mL as maximum final concentrations of the EO in the reaction mix. At such concentrations you should not have problems in dissolving the EO…. Another possibility is to use DMSO at 60% or higher to solubilize the EO: we have been using this solvent for another set of Eos (unpublished data) because it performed better than ethanol, but this depends also, as I said, on the specific type and composition of the EO… |
2楼2016-04-18 20:00:52