Method for enriching and purifying saccharide binding protein
A protein-binding and enrichment technology, applied in the field of enrichment and purification of sugar-binding proteins, can solve the problems of complex method, low recovery rate, inconvenient use, etc., and achieve the effect of simple connection method and low cost
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Embodiment 1
[0073] The implementation steps of embodiment 1 are as follows:
[0074] 1) Coupling of sugar and micro-nano magnetic particles (1) Activation of coupling agent:
[0075] ① Take the coupling agent 4-hydroxyphenylvaleric acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide according to the ratio of 1:5:5, and dissolve them in organic solvent respectively. As the solvent, ethyl acetate, dimethylformamide (DMF) and the like can be used.
[0076] ② First mix 4-hydroxyphenylvaleric acid and N-hydroxysuccinimide and dissolve them all; then add dicyclohexylcarbodiimide and mix; use ethyl acetate to dissolve to 25ml, so that the concentrations of the three are respectively 20mM, 100mM and 100mM.
[0077] ③Protected with nitrogen gas, incubated at 37°C for 12 hours, a white precipitate formed.
[0078] (2) Pretreatment of micro-nano magnetic particles derivatized with amino groups: take 15.58mg / ml of micro-nano magnetic particles derivatized with amino groups in a 5ml centrifuge ...
Embodiment 2
[0097] The implementation steps of embodiment 2 are as follows:
[0098] 1) Coupling of sugar and micro-nano magnetic particles (1) Activation of coupling agent:
[0099] ① Dissolve the coupling agent 4-hydroxybenzamide or 4-hydroxybutyric acid hydrazine in an organic solvent to prepare a 20-50mol / L coupling agent solution. The organic solvent can adopt dimethylformamide (DMF) or ethyl acetate etc.
[0100] ② Adding micro-nano magnetic particles derivatized with oxirane groups into the coupling agent solution at a ratio of 1:10.
[0101] ③ Incubate at room temperature for 3 hours.
[0102] (2) Cleaning: Magnetic separation, discarding the supernatant. Wash 3 times with ethanol and then 3 times with sodium acetate wash buffer, pH 5.6.
[0103] (3) Coupling: add 2ml of 100mM D-mannose solution prepared with pH 5.6 sodium acetate buffer, incubate at 37°C for 12 hours, then the sugar is derivatized by linking to the oxirane group On the micro-nano magnetic particle, the micro-n...
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