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Affinity chromatograph filling and producing process and application thereof

A chromatographic packing and affinity technology, applied in the field of affinity chromatography packing and its preparation, can solve the problems of difficulty in source and high cost, and achieve the effects of easy availability of raw materials, high safety and low price

Inactive Publication Date: 2006-08-02
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They tend to be highly selective, but are costly and difficult to source

Method used

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  • Affinity chromatograph filling and producing process and application thereof
  • Affinity chromatograph filling and producing process and application thereof
  • Affinity chromatograph filling and producing process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Preparation of affinity chromatography filler and affinity chromatography column with silica gel as carrier and kanamycin as ligand

[0033] 1. Preparation of affinity chromatography filler with silica gel as carrier and kanamycin as ligand

[0034] Take 1.5 g of silica gel with a particle size of 5 μm and a pore size of 300 Å, put it in a round bottom flask, add 100 mL of 10% HCl solution, soak for 18 hours, reflux at 120 ° C for 8 hours, and wash with deionized water twice until there is no Cl - (Washing solution was added dropwise with AgNO 3 until no turbidity), and dry at 120°C. Add 1.5 g of pretreated silica gel to a 100 mL round-bottomed flask, vacuumize under an oil bath at 200° C., and maintain it for 3 h. After cooling, add 50mL of 5% 3-glycidoxypropyltrimethoxysilane (GPS) toluene solution, reflux at 120°C for 8h, wash with hot methanol for 3 times, use methanol as solvent for Soxhlet extraction for 12h, and vacuum dry at room temperature . Tak...

Embodiment 2

[0039] Embodiment 2, isolate lysozyme

[0040] 1. Chromatographic retention behavior of lysozyme on blank column and affinity column

[0041] Connect the affinity chromatography column or blank column to the high performance liquid chromatography, and the sample is lysozyme. Mobile phase A is 50mM potassium phosphate buffer + 100mM NaCl (pH 7.0), and B is 50mM potassium phosphate buffer + 500mM NaCl (pH 7.0). Flow rate: 0.5mL / min; gradient conditions: 0-15min A solution, 15-30min B solution, 30-45min A solution; detection wavelength 280nm. The affinity chromatographic column with silica gel as carrier, kanamycin as ligand and blank column with no ligand bound to lysozyme were compared. The results are shown in Figure 1, which shows that lysozyme is not retained on the blank column, but can be retained on the affinity column and is eluted again when it is changed to solution B, indicating the affinity of the bonded kanamycin ligand The column has a specific effect on lysozym...

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Abstract

The present invention discloses an affinity chromatographic packing, its preparation method and application. Said packing is formed from solid-phase carrier and ligand connected with said solid-phase carrier, in which the described ligand is kanamycin. Its preparation method includes the following steps: using solid-phase carrier and kanamycin as raw material according to weight portion ratio of 2:1-15:1, utilizing amino-group of kanamycin and making it bonded on said solid-phase carrier to obtain the invented affinity chromatographic packing. Said affinity chromatographic packing can be used for separating, purifying, analyzing and determining lysozyme contained in various materials and systems.

Description

technical field [0001] The invention relates to an affinity chromatographic filler and its preparation method and application, in particular to the application of the affinity chromatographic filler in separating and purifying lysozyme. Background technique [0002] Lysozyme (lysozyme) is a basic globulin composed of 129 amino acid residues, with a molecular weight of 14.3KD. Because lysozyme has antibacterial, antiviral, and antitumor effects, it is widely used in many fields such as food, medical treatment, and bioengineering. Therefore, the research on the isolation and purification of lysozyme has attracted increasing attention. In previous studies on the preparation technology of lysozyme, methods such as salting out, gel filtration, ion exchange chromatography and affinity chromatography have all been adopted. Among them, affinity chromatography utilizes the specific affinity between the enzyme and its ligand, can effectively refine the target enzyme, and the steps ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/60G01N30/80G01N30/88
Inventor 刘阳李克安刘锋
Owner PEKING UNIV
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