Preparation method and application of metal chelating agarose gel

An agarose gel and metal chelation technology, which is applied in the field of protein chemistry, can solve the problems of general agarose gel metal chelation ability and cannot meet the requirements of protein purification, etc., and achieves strong binding ability, strong chelating ability, Easy to zoom in effect

Inactive Publication Date: 2012-05-02
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent application "Preparation method of tetraligand metal chelation chromatography packing ethylenediamine diacetic acid agarose gel" (application number CN200910196910.0) discloses a method using agarose combined with ethylenediamine diacetic acid The gel is used to chelate metal ions. The metal chelating ability of the agarose gel prepared by this method is average, and there are only 4 ligands, which still cannot meet the current protein purification requirements.

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  • Preparation method and application of metal chelating agarose gel
  • Preparation method and application of metal chelating agarose gel
  • Preparation method and application of metal chelating agarose gel

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Embodiment 1

[0021] (1) Activation: mix agarose gel (Sepharose-6B), dimethyl sulfoxide, epichlorohydrin and 0.8mol / L sodium hydroxide solution in a volume ratio of 1:1:0.5:4, then add after adding Sepharose 2% (w / v) NaBH 4 , placed on a magnetic stirrer and stirred slowly, then added 2mol / L NaOH and 2 to 3 times the volume of the agarose gel, and then added 2 to 3 times the volume of epichlorohydrin, (adding in portions within 10 hours under slow stirring , each interval of 2 hours), slowly stirred overnight at 27-35°C and then overnight, then washed with water, washed with dilute acetic acid, washed with water, and finally vacuum-filtered and dried the colloid with a Buchner funnel to obtain dried epoxy-activated agarose ;

[0022] (2) Coupling: put the blotted epoxy-activated agarose into a conical flask, add 2ml 2mol / L sodium carbonate, 0.15EDDS-Na 3 , 1.5mg NaBH 4 , stirred slowly at 50 °C for 12 hours. The Sapharose-6B connected with EDDS was vacuum-filtered and dried on a Buchner...

Embodiment 2

[0025] (1) Activation: mix agarose gel (Sepharose-6B), dimethyl sulfoxide, epichlorohydrin and 0.8mol / L sodium hydroxide solution in a volume ratio of 1:1:0.5:4, then add after adding Sepharose 2% (w / v) NaBH 4 , placed on a magnetic stirrer and stirred slowly, then added 2mol / L NaOH and 2 to 3 times the volume of the agarose gel, and then added 2 to 3 times the volume of epichlorohydrin, (adding in portions within 10 hours under slow stirring , each interval of 2 hours), slowly stirred overnight at 27-35°C and then overnight, then washed with water, washed with dilute acetic acid, washed with water, and finally vacuum-filtered and dried the colloid with a Buchner funnel to obtain dried epoxy-activated agarose ;

[0026] (2) Coupling: put the blotted epoxy-activated agarose into a conical flask, add 2ml 3mol / L sodium carbonate, 0.2g EDDS-Na 3 , 2mg NaBH 4 , stirred slowly at 50 °C for 10 h. The Sapharose-6B connected with EDDS was vacuum-filtered and dried on a Buchner funn...

Embodiment 3

[0029] (1) Activation: mix agarose gel (Sepharose-6B), dimethyl sulfoxide, epichlorohydrin and 0.8mol / L sodium hydroxide solution in a volume ratio of 1:1:0.5:4, then add after adding Sepharose 2% (w / v) NaBH 4, placed on a magnetic stirrer and stirred slowly, then added 2mol / L NaOH and 2 to 3 times the volume of the agarose gel, and then added 2 to 3 times the volume of epichlorohydrin, (adding in portions within 10 hours under slow stirring , each interval of 2 hours), slowly stirred overnight at 27-35°C and then overnight, then washed with water, washed with dilute acetic acid, washed with water, and finally vacuum-filtered and dried the colloid with a Buchner funnel to obtain dried epoxy-activated agarose ;

[0030] (2) Coupling: put the blotted epoxy-activated agarose into a conical flask, add 2ml 2mol / L sodium carbonate, 0.15g EDDS-Na 3 , 2mg NaBH 4 , stirred slowly at 30 °C for 15 h. The Sapharose-6B connected with EDDS was vacuum-filtered and dried on a Buchner funn...

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Abstract

The invention discloses a preparation method and application of metal chelating agarose gel, and belongs to the field of protein chemistry. The method for preparing the metal chelating agarose gel comprises the steps of activating and coupling. An agarose chelating medium with 6 ligands is formed by connecting ethylene diamino-disuccinic acid to sepharose. The metal chelating agarose gel can be applied to the removal of metal ions such as Cu, Zn, Mn, Cd and the like in a protein solution, and has high binding ability and binding capacity, and the removal rate reaches 93 percent. The preparation process is simple, easy to scale up and convenient to use; and the metal chelating agarose gel can be better applied to the removal of the heavy metal ions in the research of plant proteins which grow in a complex heavy metal polluted environment, and can also be applied to other protein scientific research in which metal ions are required to be removed.

Description

technical field [0001] The invention relates to the field of protein chemistry, in particular to a method for preparing metal-chelating agarose gel and its application. Background technique [0002] In the thermodynamic and kinetic analysis of metal-binding proteins, it is often necessary to apply to metal-free proteins. In ESI-MS protein profiling analysis, the presence of metal ions in the protein will lead to signal suppression and affect the analysis results; in the differential proteomics study of copper-binding proteins under copper stress, if there is excessive copper accumulation in the cell, the protein The copper-binding site in the protein will bind to the copper that enters the cell, resulting in some copper-binding proteins that cannot be isolated or are incompletely isolated. Therefore, before immobilized metal affinity chromatography, it is very important to remove metal ions in the protein solution, which can greatly increase the purification efficiency of c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/12C07K1/22
Inventor 沈振国宋玉峰陈亚华夏妍王桂萍张红晓
Owner NANJING AGRICULTURAL UNIVERSITY
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