Preparation method and application of purified avidin medium based on 6B agarose microsphere

A technology of agarose microspheres and avidin, applied in the field of protein purification, can solve the problems of low purity and purification efficiency, difficult purification of avidin, etc., to reduce non-specific binding, improve chemical stability and tolerance, The effect of high ligand density

Inactive Publication Date: 2019-08-02
武汉菲恩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the problems that existi

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  • Preparation method and application of purified avidin medium based on 6B agarose microsphere
  • Preparation method and application of purified avidin medium based on 6B agarose microsphere

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Embodiment 1

[0028] This embodiment provides a method for preparing a purified avidin medium based on 6B agarose microspheres, comprising the following steps:

[0029] (1) Cyanogen bromide and 6B agarose microspheres are mixed in the sodium carbonate buffer solution of 0.1mol / L, and the mass ratio of cyanogen bromide, 6B agarose microspheres and sodium carbonate buffer solution three is 0.1: 1:5, shaking and reacting in a shaker at 20°C for 30 minutes to obtain activated agarose microspheres.

[0030] Among them, 6B agarose microspheres means that the content of agarose accounts for 6% of the total mass. Agarose is rich in hydroxyl groups and has good biocompatibility. It is the most widely used matrix synthesis material in the process of separation and purification. ; Cyanogen bromide can quickly bind to the hydroxyl group of agarose under alkaline conditions.

[0031] (2) Wash the activated agarose microspheres obtained in step (1), add a PBS solution (phosphate buffered saline) with a ...

Embodiment 2

[0036] This embodiment provides a method for preparing a purified avidin medium based on 6B agarose microspheres, comprising the following steps:

[0037] (1) Cyanogen bromide and 6B agarose microspheres are mixed in the sodium carbonate buffer solution of 0.1mol / L, and the mass ratio of cyanogen bromide, 6B agarose microspheres and sodium carbonate buffer solution three is 0.1: 1:5, shaking and reacting in a shaker at 20°C for 30 minutes to obtain activated agarose microspheres.

[0038] Among them, 6B agarose microspheres means that the content of agarose accounts for 6% of the total mass. Agarose is rich in hydroxyl groups and has good biocompatibility. It is the most widely used matrix synthesis material in the process of separation and purification. ; Cyanogen bromide can quickly bind to the hydroxyl group of agarose under alkaline conditions.

[0039] (2) Wash the activated agarose microspheres obtained in step (1), add a PBS solution (phosphate buffered saline solution...

Embodiment 3

[0044] This embodiment provides a method for preparing a purified avidin medium based on 6B agarose microspheres, comprising the following steps:

[0045](1) Cyanogen bromide and 6B agarose microspheres are mixed in the sodium carbonate buffer solution of 0.2mol / L, and the mass ratio of cyanogen bromide, 6B agarose microspheres and sodium carbonate buffer solution three is 0.2: 1.5:3, shaking and reacting in a shaker at 37°C for 30 minutes to obtain activated agarose microspheres.

[0046] (2) The activated agarose microspheres obtained in the cleaning step (1) are added with a concentration of 1% butanediamine in PBS solution, and the mass ratio of activated agarose microspheres and butanediamine in PBS solution is 1:3, shaking in a shaker at 30°C for 1.5 hours to generate amino-modified agarose microspheres.

[0047] (3) Mix amino-modified agarose microspheres with diiminobiotin, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and diiminobiotin, 1-ethyl The mass ratio of -(3...

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Abstract

The invention provides a preparation method of a purified avidin medium based on a 6B agarose microsphere. The preparation method comprises the following steps of: 1) activating the 6B agarose microsphere through cyanogen bromide to form activated agarose microsphere, 2) allowing the activated agarose microsphere and an amino compound to give a coupling reaction to form an amino modified agarose microsphere, 3) allowing the amino modified agarose microsphere and amino biotin capable of being specifically bound with avidin to give a coupling reaction under the action of a coupling agent, and 4)adding into a PBS (phosphate buffer solution) for flushing to form the purified avidin medium after the reaction is finished. According to the preparation method, the surface of 6B agarose activatedby cyanogen bromide is subjected to amination modification by the stable amino compound; chemical stability and tolerance of a product are improved; an application scope of the agarose microsphere isexpanded; at the same time, the amino biotin capable of being dissociated sufficiently in a weakly acidic environment is adopted as a ligand for specific binding of the avidin; and the purity and purification efficiency of the avidin are improved.

Description

technical field [0001] The invention belongs to the technical field of protein purification, and in particular relates to a preparation method and application of a purified avidin medium based on 6B agarose microspheres. Background technique [0002] With the rapid development of the modern biomedical industry, the performance of biological materials is required to be gradually improved; among them, the purified avidin medium prepared from agarose is widely used in protein separation engineering, and the performance of the medium directly determines the efficiency of separation and purification. [0003] Agarose, as a kind of agarose microspheres rich in hydroxyl groups, especially the hydroxyl groups activated by cyanogen bromide, has the ability to efficiently bind proteins or amino substances, and is the most widely used matrix synthesis material in the process of affinity purification. However, since ordinary cyanogen bromide-activated agarose microspheres can only bind ...

Claims

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Application Information

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IPC IPC(8): B01J20/30B01J20/24B01J20/28B01D15/34B01D15/38C07K1/22
CPCB01D15/3804B01J20/24B01J20/28021B01J20/285B01J2220/4825C07K1/22
Inventor 董垚
Owner 武汉菲恩生物科技有限公司
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