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DEAE dextran-modified agarose gel-based chromatography medium and preparation method and application thereof

A technology of diethylaminoethylated dextran and agarose gel, which is applied in the field of protein chromatographic separation, can solve the problems of limited improvement, complex synthesis process, etc., and achieves convenient sterilization, good biocompatibility, and ease of use. effect of regeneration

Active Publication Date: 2015-12-30
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, the synthesis process is relatively complicated; on the other hand, although the ion-exchange chromatographic medium modified by neutral low-molecular-weight dextran can improve the adsorption capacity and mass transfer rate of proteins to a certain extent, the degree of improvement is very limited.

Method used

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  • DEAE dextran-modified agarose gel-based chromatography medium and preparation method and application thereof
  • DEAE dextran-modified agarose gel-based chromatography medium and preparation method and application thereof
  • DEAE dextran-modified agarose gel-based chromatography medium and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] (1) Take 1g of agarose gel drained by the G3 funnel and put it into a 50mL Erlenmeyer flask, add 2mL of dimethyl sulfoxide and 1.3mL of epichlorohydrin in sequence, and mix in a shaker at 25°C and 170rpm 0.5h. Then add 2mL of sodium hydroxide (1mol / L) and react for 2.5h. Rinse repeatedly with deionized water until the cleaning solution is washed with phenolphthalein–Na 2 S 2 o 3 The solution does not change color after detection, and the agarose gel medium with active epoxy groups on the surface is prepared. The epoxy group modification density of the agarose gel medium with active epoxy groups on the surface is 51mmol / L.

[0039] (2) Add 1 g of agarose gel medium with active epoxy groups on the surface to 1 mL of DEAEDextran (Mw500kDa) aqueous solution (200 mg / mL) drained with a G3 funnel, and place it in a constant temperature shaker at 20 ° C, 120 rpm Diffuse for 1 hour to fully diffuse DEAEDextran into the media pores, then add 2 mL of sodium hydroxide (0.1mol / ...

Embodiment 2

[0041] Add 3 g of the agarose gel medium with active epoxy groups on the surface of the G3 funnel drained in step (1) of Example 1 to 3 mL of DEAEDextran (Mw500kDa) aqueous solution (300 mg / mL), and place at a constant temperature of 25 ° C. In a shaker, diffuse at 170rpm for 2.5h to fully diffuse DEAEDextran into the medium pores, add 3mL of sodium hydroxide (1mol / L), react at 25°C and 170rpm for 48h to couple DEAEDextran to the agarose gel, and use Rinse with deionized water repeatedly until the cleaning solution is detected by phenolphthalein and does not change color, then place the medium in 0.5g / L sodium borohydride solution, react at room temperature for 12 hours to reduce the residual epoxy groups on the surface of the medium, and use deionized water again to reduce the medium After repeated washing, the DEAEDextran grafted agarose gel medium with the ion exchange capacity of structural formula (I) of 90 mmol / L was obtained.

Embodiment 3

[0043] Add 5 g of the agarose gel medium with active epoxy groups on the surface of the G3 funnel drained in step (1) of Example 1 to 5 mL of DEAEDextran (Mw500kDa) aqueous solution (600 mg / mL), and place at a constant temperature of 25 ° C In a shaker, diffuse at 200rpm for 2.5h to fully diffuse DEAEDextran into the medium pores, add 5mL of sodium hydroxide (1mol / L), react at 25°C and 170rpm for 60h to couple DEAEDextran to the agarose gel, and use Rinse with deionized water repeatedly until the cleaning solution is detected by phenolphthalein and does not change color, then place the medium in 0.5g / L sodium borohydride solution, react at room temperature for 12 hours to reduce the residual epoxy groups on the surface of the medium, and use deionized water again to reduce the medium Washing was repeated to prepare a DEAEDextran grafted agarose gel medium with the ion exchange capacity of structural formula (I) of 199 mmol / L.

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Abstract

The invention relates to a DEAE dextran-modified agarose gel-based chromatography medium and a preparation method and application thereof. The method includes the steps that first, the molecular weight of DEAE Dextran for grafting is selected; next, the concentration of a DEAE Dextran aqueous solution is controlled; then, the time of diffusion before DEAE Dextran coupling is determined; finally, the reaction time of coupled DEAE Dextran and the concentration and volume of sodium hydroxide are determined. The DEAE dextran-modified agarose gel-based chromatography medium has a strong adsorption property for protein under different modification densities, shows a high adsorption capacity and a high adsorption rate and improves separation efficiency. The medium is convenient to clean and degerm, easy to regenerate and good in biocompatibility. The preparation method is simple and harmfulless and has broad application prospects in efficient and rapid separation and purification of protein.

Description

technical field [0001] The invention relates to a chromatographic medium based on agarose gel modified by diethylaminoethylated dextran and its preparation method and application. It can significantly improve the protein adsorption capacity and mass transfer rate, and belongs to the protein in the field of biotechnology. Chromatographic separation techniques. Background technique [0002] Diethylaminoethylated dextran (DEAEDextran) is a long-chain cationic polyelectrolyte modified with diethylaminoethyl (DEAE) on dextran. DEAEDextran is positively charged and can reversibly adsorb negatively charged substances. DEAEDextran has good biocompatibility and is mostly used in gene transfection and sustained sustained release of proteins. In recent years, researchers have found that DEAEDextran can also be applied to cell immobilization in biosensors. [0003] DEAEDextran has a larger molecular weight and more hydroxyl groups, and it is easy to obtain a DEAEDextran-grafted chrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/291B01J20/286B01J20/26B01J20/30C08B37/12C08B37/02C07K1/22
Inventor 余林玲白姝龚玲莉孙彦
Owner TIANJIN UNIV
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