A method for separating and purifying cobra venom neurotoxin in combination with ion exchange and hydrophobic chromatography
A technique for separation and purification of cobra venom, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of long separation and purification cycle and complicated operation steps, and achieve shortened cycle, simplified operation steps and high yield high effect
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Embodiment 1
[0031] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:
[0032] (1) Weigh 4.5g of crude cobra venom, add it to 0.045L water or 0.03M phosphate buffer with pH 6.0, stir to dissolve, centrifuge at 10000rpm at 4°C, discard the precipitate, and use 45μm PES to supernatant Microporous filter membrane is filtered, filters, and needs testing solution A, for subsequent use;
[0033] (2) Need testing solution A is purified by ion-exchange chromatography, the medium of ion-exchange chromatography is CM SepharoseFast Flow, with the 0.03M phosphate buffer of pH 6.0 as mobile phase A, to comprise 1M sodium chloride The 0.03M phosphate buffer with pH 6.0 is the mobile phase B. First, use 2 column bed volumes of mobile phase B for activation, then use 3 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution. The steps of gradient elution are: A:B volume ratio is 100%:0%, A:B volume rat...
Embodiment 2
[0037] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:
[0038] (1) Weigh 4g of crude cobra venom, add it to 0.04L water or 0.01M phosphate buffer with pH 5.0, stir to dissolve, centrifuge at 8000rpm at 0°C, discard the precipitate, and use 45μm PES to micronize the supernatant. Pore filter membrane is filtered, filters, and needs testing solution A, for subsequent use;
[0039](2) Purify need testing solution A by ion exchange chromatography, the medium of ion exchange chromatography is CM SepharoseFast Flow, with the 0.01M phosphate buffer of pH 5.0 as mobile phase A, to include 0.4M sodium sulfate The 0.01M phosphate buffer with pH 5.0 is used as mobile phase B. First, activate with 1 column bed volume of mobile phase B, then use 2 column bed volumes for equilibrium elution, and then gradient elution. The steps of gradient elution are: A:B volume ratio is 100%:0%, A:B volume ratio is 90%:10%, A:B v...
Embodiment 3
[0043] The separation and purification method of cobra venom neurotoxin of the present embodiment comprises the following steps:
[0044] (1) Weigh 5g of crude cobra venom, add it to 0.05L water or 0.05M phosphate buffer solution with pH 7.0, stir to dissolve, centrifuge at 12000rpm at 8°C, discard the precipitate, and use 45μm PES to micronize the supernatant. Pore filter membrane is filtered, filters, and needs testing solution A, for subsequent use;
[0045] (2) Need testing solution A is purified by ion exchange chromatography, the medium of ion exchange chromatography is CM SepharoseFast Flow, with the 0.05M phosphate buffer of pH 7.0 as mobile phase A, to comprise 0.4M sodium phosphate The 0.05M phosphate buffer with pH 7.0 was used as the mobile phase B, firstly activated with 3 column bed volumes of mobile phase B, then used 4 column bed volumes of mobile phase A for equilibrium elution, and then gradient elution, The steps of gradient elution are: A:B volume ratio ...
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