Affinity chromatography column and preparation method and application thereof

A chromatographic column and affinity technology, applied in the field of separation and purification of biological macromolecules

Active Publication Date: 2021-04-27
杭州华安生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned affinity chromatography column effectively alleviates the clumping / coagulation of the cyanogen bromide-activated agarose chromatography medium caused by the easy precipitation of the ligand after denaturant treatment in the preparation process of the existing cyanogen bromide-activated agarose affinity chromatography column. block problem

Method used

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  • Affinity chromatography column and preparation method and application thereof
  • Affinity chromatography column and preparation method and application thereof
  • Affinity chromatography column and preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0036] According to one aspect of the present invention, a kind of preparation method of above-mentioned affinity chromatography column, described preparation method comprises the following steps:

[0037] The denaturant-treated biological macromolecules containing amino ligands, cyanogen bromide-activated agarose chromatography packing materials and surfactants were coupled and incubated in the empty column of the chromatography column to obtain a reaction column with a stationary phase. The reaction column is washed and blocked sequentially to obtain an affinity chromatography column.

[0038] The preparation method of the affinity chromatography column provided by the invention, the preparation method is that the biomacromolecule containing the amino ligand after denaturant treatment, cyanogen bromide activated agarose chromatography filler and surfactant are mixed in the column space A coupling incubation reaction is carried out in the column to obtain a reaction column wi...

Embodiment 1

[0046] 1. Prepare the solution:

[0047] 1. Ligand solution containing amino groups: the denaturant used in this embodiment is 4M urea, the amount of protein is 2 mg, and the concentration is 1 mg / ml.

[0048] The specific preparation method is as follows: the protein concentration of the amino ligand to be coupled is 2 mg / ml, the dosage is 2 ml, the protein buffer solution is 4M urea, pH 7.4.

[0049] 2. 1mM hydrochloric acid solution with pH 3.0: Take 0.1mL of concentrated hydrochloric acid, add 1.1mL of UP water, mix well, and prepare 1M dilute hydrochloric acid. Then take 1mL from 1M dilute hydrochloric acid, dilute it to 1L and make it into 1mM dilute hydrochloric acid, and measure the pH to be 3.0.

[0050] 3. 0.1M Tris-HCl blocking solution with pH 8.0: Weigh 6.057g Tris and dissolve in 400mL of UP water, adjust the pH to 8.0 with HCl and dilute to 500mL.

[0051] 4. Circulating buffer solution A: weigh 4.1g of anhydrous CH 3 Dissolve COONa, 14.61g of NaCl in 490mL o...

experiment example 1

[0066] In order to show that the affinity chromatography column prepared by this application can effectively alleviate the precipitation of the ligands after the denaturant treatment in the preparation process of the existing cyanogen bromide-activated agarose affinity chromatography column, which leads to the cyanogen bromide-activated agarose layer. In order to solve the problem of agglomeration / blocking of the analysis medium, the affinity chromatography column prepared in Example 1 and Comparative Example 1 is now used for chromatographic separation of serum and purified IgG antibodies, and the separated and purified serum and IgG antibodies are ELISA detection, as follows:

[0067] (1) Sample acquisition method: Provide an amino-containing ligand with a protein concentration of 1 mg / ml, take 0.5 ml of sutra and immune adjuvant (complete adjuvant for the first immunization of rabbits, and incomplete adjuvant for subsequent immunizations) in a volume ratio of 1 :1 After mix...

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Abstract

The invention provides an affinity chromatography column as well as a preparation method and application thereof, and relates to the technical field of separation and purification of biomacromolecules. The stationary phase of the affinity chromatography column is mainly prepared by coupling incubation reaction of amino ligand-containing biomacromolecules treated by a denaturing agent, cyanogen bromide activated agarose chromatography filler and a surfactant. According to the affinity chromatography column, the surfactant is added into the stationary phase, so that coupling of the amino ligand-containing biomacromolecules treated by the denaturing agent and the cyanogen bromide activated agarose chromatography filler can be effectively promoted, and the problems of agglomeration and caking of a cyanogen bromide activated agarose chromatography medium caused by easy precipitation of ligand treated by a denaturing agent in the preparation process of the existing cyanogen bromide activated agarose affinity chromatography column are effectively relieved.

Description

technical field [0001] The invention relates to the technical field of separation and purification of biological macromolecules, in particular to an affinity chromatography column and its preparation method and application. Background technique [0002] At present, other ligands containing amino groups, such as proteins, polypeptides and nucleic acids, are coupled to purification medium fillers on the market, mainly by using cyanogen bromide to activate agarose chromatography medium. Cyanogen bromide-activated agarose chromatography medium is an activated intermediate filler, which generates activated cyanate groups through the reaction of cyanogen bromide and hydroxyl groups on the agarose, and the activated cyanate groups combine with proteins, Molecules with amino groups such as polypeptides and amino acids are coupled, and the coupling reaction can be carried out in aqueous or organic phases. [0003] The cyanogen bromide-activated fillers currently on the market still ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/20B01D15/38C07K16/06C07K1/22
CPCB01D15/206B01D15/3804C07K16/065C07K1/22
Inventor 徐晓勇田向荣张丰
Owner 杭州华安生物技术有限公司
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