Immobilized metal ion affinity chromatography filler, chromatographic column and preparation method thereof

A technology for immobilizing metals and chromatographic columns, applied in the field of chromatography, can solve the problems of low selectivity, poor chelating performance, high price, etc., and achieve good physical and chemical stability, high repeatability and yield, and low preparation cost. Effect

Active Publication Date: 2021-10-15
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the solid phase matrix of commercial IMAC products is usually resin microspheres or magnetic particles, so that the process of loading, washing and elution can be carried out by means of centrifugation or magnetic adsorption. Iminodiacetic acid (IDA), aminotriacetic acid (NTA ) and ethylenediamine (TED) are representative chelating ligands for immobilizing metal ions, and their carboxyl and amine groups can bind Al 3+ , Fe 3 + , Ga 3+ 、Ni 2+ and other metal ions are chelated on the surface of the solid phase matrix, but the chelation performance of these combinat...

Method used

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  • Immobilized metal ion affinity chromatography filler, chromatographic column and preparation method thereof
  • Immobilized metal ion affinity chromatography filler, chromatographic column and preparation method thereof
  • Immobilized metal ion affinity chromatography filler, chromatographic column and preparation method thereof

Examples

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Embodiment 1

[0037] Example 1 Preparation of ATP-modified immobilized metal ion affinity capillary monolithic column

[0038] The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by a one-step reaction method in the present invention specifically comprises the following steps:

[0039] S1. Add 740 microliters (about 1000 mg) of potassium water glass (modulus 3.3, Baume degree 40) to the micro-reaction flask, and slowly add 6 microliters (about 6 mg) of γ-glycidyl ether oxypropylene under stirring at room temperature Trimethoxysilane (GLYMO, Cas number: 2530-83-8), and continued to stir well at room temperature for 30 minutes.

[0040] S2. Weigh 7.5 mg of adenosine triphosphate disodium, dissolve it in 160 microliters of deionized water, slowly add it to the reaction solution in step S1, and continue to stir fully at room temperature for 30 minutes.

[0041] S3. Take 60 microliters (about 68 mg) of formamide, mix it with 40 microliters of deionized water, sl...

Embodiment 2

[0046] Example 2 The ability of the ATP-modified immobilized metal ion affinity capillary monolithic column prepared in Example 1 of the present invention to enrich phosphorylated peptides was comprehensively investigated.

[0047] The ATP-modified immobilized metal ion affinity capillary monolithic column prepared in Example 1 of the present invention was used for the enrichment of phosphorylated peptides obtained by treating standard phosphorylated protein α-casein or BSA with trypsin. The enrichment performance was examined from the aspects of site coverage, detection limit and selectivity.

[0048]2mg / ml BSA dissolved in 50mM ammonium bicarbonate solution was added to DTT to 10mM and heated at 56°C for 30min, then added iodoacetamide to 40mM and incubated in the dark for 40min to break the disulfide bond. Take 1 mg of standard phosphorylated protein α-casein and 1 mg of BSA that breaks disulfide bonds, dissolve in 1 ml of 50 mM ammonium bicarbonate, and treat with 20 μg tr...

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Abstract

The invention discloses an immobilized metal ion affinity chromatography filler, a chromatographic column and a preparation method thereof. The method comprises the following steps: mixing and stirring potash water glass, gamma-glycidyl ether oxypropyl trimethoxy silane and water-dissolved adenosine disodium triphosphate, then adding water-dissolved formamide, stirring to obtain a reaction solution, reacting and curing to obtain a solid material, and then washing to obtain the immobilized metal ion affinity chromatographic column filler. The ATP modified immobilized metal ion affinity capillary monolithic column prepared through a one-step reaction method is simple and rapid in preparation step, high in repeatability and yield and low in preparation cost. The product can be obtained by uniformly mixing the raw materials and then carrying out one-time baking reaction, and is good in physical and chemical stability, and has relatively good reproducibility after being used for multiple times.

Description

technical field [0001] The invention belongs to the field of chromatography, and in particular relates to an immobilized metal ion affinity chromatography filler, a chromatography column and a preparation method thereof. Background technique [0002] Immobilized metal ion affinity chromatography (IMAC) is used for affinity purification of proteins, enzymes, polypeptides and amino acids that have the ability to bind to metal ions. The principle is to enrich and purify specific proteins / peptides by utilizing the electrostatic interaction between metal ions on solid-phase carriers and some amino acids or chemical modification groups exposed by proteins / peptides. After 30 years of development , has gradually become one of the most effective techniques for separating and purifying biomolecules such as proteins. [0003] The core of immobilized metal ion affinity chromatography (IMAC) lies in the selection and preparation process of solid phase matrix, ligands and metal ions. The...

Claims

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Application Information

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IPC IPC(8): B01J20/285B01J20/283B01D15/38B01J20/30
CPCB01J20/285B01J20/283B01D15/3804
Inventor 张文洋晏石娟黄文洁吴绍文殷志斌孔谦
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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