Method for rapidly screening natural products with transmembrane transport activity at high throughput
A natural product, high-throughput technology, applied in the fields of resistance to vector-borne diseases, measurement devices, and material analysis through optical means, which can solve problems such as time-consuming, sophisticated equipment requirements, and cumbersome screening technology steps
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Embodiment 1
[0036] A method for rapid high-throughput screening of natural products with transmembrane transport activity, the steps are as follows:
[0037] Step 1. Add 1 mL of EYPC solution (20 mg / mL) dissolved in dry chloroform into a round-bottomed flask, remove the mixed solvent under reduced pressure at 40 °C, and add 1 mL of a buffer solution containing HPTS (1 mmol / L) after vacuum drying for 24 h. NaCl 100 mM, HEPES 10 mM, pH=7.0), the vesicle fluid was rapidly cooled and warmed 10 times, that is, frozen in liquid nitrogen for 1 min, and heated in a 37°C water bath for 1.5 min. The phospholipid unilamellar vesicle suspension was extruded through a filter membrane with a pore size of 200 nm to prepare a phospholipid unilamellar vesicle suspension with a diameter of about 200 nm, and the phospholipid unilamellar vesicle suspension was passed through Sephadex G-50 column to remove unembedded fluorescent dye to prepare 5 mL of vesicle stock solution with a final concentration of 6.5 mM...
Embodiment 2
[0046] Step 1. Add 1 mL of EYPC solution (20 mg / mL) dissolved in dry chloroform into a round-bottomed flask, and remove the mixed solvent under reduced pressure at 40°C. After vacuum drying for 24 hours, add 1 mL, 1 mmol / L of buffer solution containing carboxyfluorescein (CF) fluorescent probe (NaCl 100 mM, HEPES 10 mM, pH=7.5), rapidly cool the vesicle liquid and return to temperature 10 times, that is, in the liquid Freeze in nitrogen for 1 min and heat in a water bath at 37°C for 1.5 min. Extrusion through a filter membrane to prepare unilamellar vesicles about 200 nm in diameter, passing the vesicle suspension through Sephadex G-50 column to remove unembedded CF fluorescent dye to prepare 5 mL of vesicle stock solution with a final concentration of 6.5 mM.
[0047] Step 2: Dissolve 150 μL of the above-mentioned vesicle stock solution containing the CF fluorescent probe in 14.85 mL of NaCl (NaCl 100 mM, HEPES 10 mM, pH=7.5) buffer solution respectively. Take 200 μL of th...
Embodiment 3
[0054] like Figure 4 Shown, a method for rapid high-throughput screening of natural products with transmembrane transport activity, compared to Example 1:
[0055] In step (2), the outside of the vesicle is a buffer solution of MCl 100mM containing cations, pH=8.0.
[0056] Such methods are used to screen compounds with cationic transmembrane transport activity, where the cation M is Li + , Na + , K + , Rb + , Cs + , Ca 2+ , Mg 2+ , H + and NMDG + one or more of them.
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