Preparation method of snail respiratory protein
A snail and protein technology, applied in the fields of medicine extraction and biology, can solve the problems of slow gel filtration purification, many impurities in hemocyanin, and difficulty in extracting hemolymph.
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Embodiment 1
[0021] Take the snails in batches on a glass plate, mechanically crush and remove the shells, intercept the required tissues, and mechanically squeeze the Oncomelania hemolymph to a centrifuge tube; add Tris-HCl pH7.0 buffer solution, mechanically squeeze and stir to fully dissolve, and centrifuge at 8000g After 20 minutes, discard the precipitate; add ammonium sulfate to the supernatant to a saturation of 33%, let it stand at 4°C for 2 hours, and centrifuge to remove the supernatant to obtain a crude hemocyanin blue precipitate.
[0022] The hemocyanin precipitate was redissolved with the aforementioned Tris-HCl buffer, dialyzed overnight at 4°C; transferred to a centrifuge tube at 180,000g, centrifuged at 4°C for 3.5h, and the supernatant was discarded to obtain a blue protein precipitate again, which was dissolved in 0.05M Tris -HCl (pH7.0, containing 5mM CaCl 2 , 5 mM MgCl 2 ) in the buffer solution. The protein purity is above 95%.
Embodiment 2
[0024] Take snails in batches on a glass plate, mechanically crush and remove the shells, intercept the required tissues, mechanically squeeze and collect Oncomelania hemolymph into a centrifuge tube; mechanically squeeze and collect hemolymph, and centrifuge at 10,000g for 50 minutes to remove impurities such as remaining tissue fragments; add Tris-HCl pH 7.0 buffer was used to dissolve hemolymph, and ammonium sulfate was added to reach a saturation of 43%, and the protein was precipitated at 8°C for 3 hours, and the supernatant was removed by centrifugation to obtain a crude blue precipitate of hemocyanin.
[0025] Hemocyanin precipitate was redissolved with the aforementioned Tris-HCl buffer, dialyzed overnight at 8°C; transferred to a centrifuge tube at 180,000g, centrifuged at 6°C for 4.5h, discarded the supernatant, and obtained blue protein precipitate again, and dissolved the precipitate in 0.08M Tris -HCl (pH7.0, containing 5mM CaCl 2 , 5 mM MgCl 2 ) in the buffer so...
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