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Preparation method of agarose gel for affinity chromatography

A kind of technology of agarose gel and affinity, which is applied in the field of preparation of agarose gel, can solve the problems of low polypeptide connection amount and high cost of resin preparation, and achieve the effect of simple preparation process, easy operation and enlarged production scale

Active Publication Date: 2017-09-26
WUXI APPTEC SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can solve the problems of high resin preparation cost and low amount of connection with polypeptide in the process of antigen antibody purification chromatography

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation method of the agarose gel that is used for affinity chromatography, the steps are as follows:

[0034] 1) Take 100ml Sepharose CL-4B (Sepharose CL-4B) and wash it with 1L pure water and transfer it to a beaker, add 0.6M150ml containing 800mg NaBH 4 Add 150ml of epichlorohydrin slowly and keep stirring overnight (for example, 16 hours) to obtain the first glue.

[0035] 2) Wash (wash) the first glue in the order of pure water, acetone, and pure water 1L each, and take 1M pH9.5NaHCO 3 Place 50ml of the solution in an ice bath, add 25ml of dipropylenetriamine, adjust the pH to 9.5, then add 1M pH9.5NaHCO 3 When the solution reaches a final volume of 100ml, add the first gel and keep stirring at room temperature overnight (for example, 16 hours) to obtain the second gel.

[0036] 3) After washing the second gel with 1L of pure water, add 300ml of 1M ethanolamine solution to the second gel, and keep stirring at room temperature for 3 hours to obtain the thi...

Embodiment 2

[0040] The preparation method of the agarose gel that is used for affinity chromatography, the steps are as follows:

[0041] 1) Wash the agarose gel CL-6B (100ml if possible) with 1L of pure water, transfer it to a beaker, and add 0.55mol / L NaBH 4 NaOH solution (where NaBH 4 The mass ratio to NaOH is 1:4, containing NaBH 4 The volume ratio of NaOH solution to agarose gel is 6:5), and then add chloroethylene oxide (the volume ratio of chloroethylene oxide to agarose gel is 6:5) and stir for 12 hours to obtain the Wash the first gel in the order of pure water, ethanol, and pure water.

[0042] 2) Under ice bath, in 0.5mol / L NaHCO of pH10 3 Add dipropylenetriamine (dipropylenetriamine and the first part of the second alkali solution to the volume ratio of Sepharose CL-6B in the range of 1:1.5) The volume ratio is 1:2.5), adjust the pH to 10, and then add 0.5mol / L NaHCO of pH10 3 After the solution reaches the final volume (the volume ratio of the final volume to the agarose...

Embodiment 3

[0046] The preparation method of the agarose gel that is used for affinity chromatography, the steps are as follows:

[0047] 1) After washing the agarose gel CL-6B (100ml for example) with 1L of pure water, transfer it to a beaker, and add 0.65mol / L NaBH 4 NaOH solution (where NaBH 4 The mass ratio to NaOH is 1:5, containing NaBH 4 The volume ratio of NaOH solution to agarose gel CL-6B is 2:1), then add epoxy chloroethylene (the volume ratio of epoxy chloroethane to agarose gel is 2:1) and stir for 20 hours , to obtain the first glue, and wash the first glue in the order of pure water, ethanol, and pure water respectively.

[0048] 2) Under ice bath, in 1.5mol / L NaHCO of pH9 3 solution (as the first part of the second alkaline solution, the volume ratio of which to Sepharose CL-6B is 1:1) by adding dipropylene triamine (dipropylene triamine and the first part of the second alkaline solution The volume ratio is 1:3), adjust the pH to 9, and then add 1.5mol / L NaHCO of pH9 ...

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PUM

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Abstract

The invention discloses a preparation method of agarose gel used for affinity chromatography. The preparation method comprises following steps: 1) agarose gel is washed, and is delivered into a container, a first alkali solution is added, and an epoxy compound is added so as to obtain a first gel via stirring, and the first gel is washed; 2) in ice bath, dipropyl triamine is added into a second alkali solution, pH value is adjusted, the second alkali solution is added, the first gel is added, and an obtained mixture is stirred so as to obtain a second gel; 3) the second gel is washed, an alcohol amine organic matter is added into the second gel for stirring so as to obtain a third gel, and the third gel is washed; 4) an iodoacetic acid solution is prepared, pH value of the iodoacetic acid solution is adjusted to 4 to 5, the third gel and EDC.HCl are added, pH valued is maintained to be 4 to 5, an obtained mixed material is stirred so as to obtain a fourth gel, and the fourth gel is washed so as to obtain the agarose gel used for affinity chromatography. Preparation processes of the preparation method are simple; operation is convenient; a problem that resin preparation cost in antigen-antibody purification processes is high is solved; polypeptide coupling efficiency and adsorption amount of protein to be separated and purified are increased; and protein production scale is enlarged.

Description

technical field [0001] The invention relates to a method for preparing an agarose gel, in particular to a method for preparing an agarose gel for affinity chromatography. Background technique [0002] Affinity chromatography is a chromatographic system that applies the principle of reversible binding between biopolymers and ligands, and binds the ligands firmly to the carrier through covalent bonds. Due to its strong hydrophilicity, stable physical and chemical properties, and loose network structure, agarose gel has almost no non-specific adsorption to proteins at a certain buffer ion concentration, and is currently widely used in affinity chromatography, especially Antigen antibody purification. For polypeptides or proteins containing thiols, the currently commercialized Thermo Scientific SulfoLink coupling resin is porous 6% cross-linked microbeaded agarose, and its immobilization capacity is 1-2 mg of polypeptide or 2 mg per 2 ml of SulfoLink coupling resin. -20mg prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/30C07K1/22C08B37/12
Inventor 李幸孙晓磊夏波
Owner WUXI APPTEC SUZHOU
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