NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and preparation method and application thereof

A matrix and aldehyde-based technology, applied in the field of NTA-based IMAC filler and its preparation, can solve the problems of high raw material costs and other issues

Active Publication Date: 2019-04-23
SEPAX TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, it is necessary to provide a kind of NTA-based IMAC filler and its preparation method and application for the high cost of raw materials for preparing NTA-based IMAC filler

Method used

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  • NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and preparation method and application thereof
  • NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and preparation method and application thereof
  • NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0029] See figure 1 , a kind of preparation method of the NTA-based IMAC filler of one embodiment of the present invention, comprises the steps:

[0030] S10. A matrix is ​​provided.

[0031] Preferably, the matrix is ​​a polymer matrix with hydroxyl groups on the surface or a polymer matrix with epoxy groups on the surface. The surface of the polymer matrix contains hydroxyl or epoxy groups, which is convenient for the next step of reaction.

[0032] Preferably, the polymer matrix is ​​selected from one of polymethacrylate microspheres and polystyrene-divinylbenzene microspheres.

[0033] S20. Constructing aldehyde groups on the surface of the substrate to obtain a substrate with aldehyde groups on the surface. As shown in formula (1):

[0034] SP-R-CHO

[0035] Formula 1)

[0036] Among them, SP represents the substrate, and R represents the connecting arm between the substrate and the aldehyde group.

[0037] Preferably, when the substrate is a polymer substrate cont...

Embodiment 1

[0067] The polymethacrylate microspheres used in this example, the model is Monomix MC30 (particle size is 30 μm, pore size is 100 nm, the surface contains a lot of hydroxyl groups, and the color is white); the manufacturer is Suzhou Saifen Technology Co., Ltd. self-made. All other raw materials are commercially available.

[0068] Weigh 100g of polymethacrylate microspheres with a balance, transfer to a 500mL three-neck flask, add 100mL of 0.6M NaOH solution (containing 2mg / mL NaBH 4 ), then add 100mL 1,4-butanediol diglycidyl ether, and react at 37°C with 100rpm mechanical stirring for 4h. Wash 3 times with deionized water until neutral. Then the solid product obtained was added to a 1000mL three-necked flask, and 300mL of 0.5M H 2 SO 4 The solution was reacted at a temperature of 50° C. with 100 rpm mechanical stirring for 2 hours. After the reaction, the reaction solution was drained with a Buchner funnel to keep the solid product, and washed with deionized water until ...

Embodiment 2

[0074] The polymethacrylate microspheres used in this example, the model is Monomix MC30-Epoxy (particle size is 30 μm, pore size is 100 nm, the surface contains epoxy groups, and the color is white); the manufacturer is Suzhou Saifen Technology Co., Ltd. Prepare yourself. All other raw materials are commercially available.

[0075] Weigh 100g of polymethacrylate microspheres with a balance, transfer to a 500mL three-necked flask, add 150mL of 0.1M sodium carbonate-sodium bicarbonate buffer solution, adjust the pH of the mixture to 10.5, then add 89.6g of glucosamine, and control the temperature 70°C, 100rpm mechanical stirring and reaction for 16h. After the reaction, use a Buchner funnel to dry it, keep the solid product, and wash it with deionized water for 5 times to obtain polymethacrylate microspheres with aldehyde groups on the surface.

[0076] The rest of the experimental steps were consistent with the corresponding experimental steps in Example 1 to obtain the NTA-b...

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Abstract

The invention relates to an NTA-based (nitrilotriacetate) IMAC (immobilized metal affinity chromatography) filler and a preparation method and an application thereof. The prepared method of the NTA-based IMAC filler includes the following steps of providing a matrix, constructing an aldehyde group on the surface of the matrix to obtain a matrix with the aldehyde group on the surface, converting the aldehyde group to alpha-aminocarboxylic acid to obtain a matrix with the alpha-aminocarboxylic acid on the surface, constructing an NTA group on the surface of the matrix having the alpha-aminocarboxylic acid on the surface to obtain a matrix with the NTA group on the surface, and chelating the NTA group with transition metal ions to obtain the NTA-based IMAC filler. With the above-mentioned preparation method of the NTA-based IMAC filler, the use of high-cost raw materials is avoided, preparation process is clean, and purification process is simple.

Description

technical field [0001] The invention relates to the technical field of fillers, in particular to an NTA-based IMAC filler and its preparation method and application. Background technique [0002] The concept of immobilized metal affinity chromatography was first proposed by Porath et al. in 1975. Its principle is based on the immobilization of transition metal ions such as Zn on a solid support. 2+ 、Cu 2+ 、Ni 2+ 、Co 2+ The affinity of histidine residues and cysteine ​​residues can be used to separate the target protein. Following the publication of the work by Porath et al., IMAC technology has been widely used in various protein and peptide purification applications. So far, the chelating ligand iminodiacetic acid (IDA) studied by Porath et al. for immobilizing metal ions on solid supports is still used by many IMAC products. In 1987, Hochuli et al. invented an improved metal chelating ligand nitrilotriacetic acid (NTA). With the rapid development of modern molecular b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/08C07K1/22
CPCB01D15/08C07K1/22
Inventor 吕小林丁忠
Owner SEPAX TECH
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