Dear Markaz,
I guess your would be interested to check out our manuscript in BMC Genomics about interaction studies of Odourant binding proteins with kairomones. Please follow the link to know more about the methods used. You can contact me in case you have any doubts.
Cheers,
Vivek Kempraj
Article Computational reverse chemical ecology: Virtual screening an...
Hello Marakaz
Use fluorescence competitive binding assays to measure the binding assay of OBP and odor substances in vitro. 1-NPN (N-phenyl-1-naphthylamine) is a good fluorescence indicator (fluorescent probe) for OBPs (OBP/PBP/CSP)
Specific steps are as follows:
(1) Sample preparation: the purified protein was soluble in 50 mm Tris - HCl buffer (pH 7.4), with the concentration of mg/ml level of mother liquor, the concentration of protein was determined by spectrophotometry. Fluorescent probe (1 - NPN) and odor material were dissolved in chromatographic grade methanol with 10 or 100 mM first as mother liquor, and diluted with methanol to 1 mM, when used as the working fluid, stored in - 20 ℃.
(2) Determination of fluorescent probes suitability: in 2 micromole protein solution, joining the 1-NPN (concentration of 1 mM) to ensure the final concentration is 0-20 micromoles solution, to determine whether this probe can be used for competitive combining to experiment, and measure dissociation constant K1 - NPN (experimental parameters setting: Ex = 337 nm, Em = 405 nm, the Ex Slit = 9 nm, Em Slit = 15 nm). If the combinations of 1 - NPN and OBPs have saturation effect and Scatchard analysis shows linear relationship, it shows that combinations of both present a combination of a single site, and can be combined for the subsequent competitive experiment.
(3) Competitive combination experiment: first, mix 1 – NPN and OBPs (both concentrations are 2 micromoles), let stand for 2 min to make a combination of both, and then measure the fluorescence intensity; Then join the final concentration of 0.25 to 0.25 micromoles (sex pheromone) or 2-20 micromoles (scent) ligands one by one and let stand for 2 min, measuring the fluorescence intensity.
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