Preparing method and application of gelatin affinity chromatography media

A chromatographic medium and affinity technology, applied in the field of preparation of gelatin affinity chromatographic medium, can solve the problems of low repeatability, difficult to scale up production, increase production cost, etc. Mass production, the effect of increasing production costs

Active Publication Date: 2015-10-21
WUHAN HUIYAN BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The disadvantage of the existing preparation technology is that the preparation method is divided into three steps of freeze-drying, the reaction and cleaning process are cumbersome and complicated, and the time is too long; the required production equipment is too much and expensive, which seriously increases the production cost; the equipment and process control is relatively strict , is only suitable for laboratory-level preparation, and it is not easy to scale up to production line scale production; the roundness of the product is poor, the particle size and pore size are too large, and the low temperature in the production process will lead to unstable product quality and low repeatability, etc.
[0006] The disadvantages of the existing separation and purification technology are that there are too many separation and purification steps, it takes too long, the activity of fibronectin is low, and the protein concentration and purity are too low

Method used

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  • Preparing method and application of gelatin affinity chromatography media
  • Preparing method and application of gelatin affinity chromatography media
  • Preparing method and application of gelatin affinity chromatography media

Examples

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preparation example Construction

[0069] Such as figure 1 As shown, a method for preparing a gelatin affinity chromatography medium includes the following steps:

[0070] 1) Weigh the CNBr-agarose affinity chromatography medium and add it to the suction filter funnel, and clean the CNBr-agarose affinity chromatography medium with the activation solution pre-cooled at 4-8°C. The volume of the activation solution is CNBr-agarose 5-10 times the volume of the affinity chromatography medium, wash for 15-30 minutes to remove the activation solution; then wash the CNBr-agarose affinity chromatography medium with the binding solution pre-cooled at 4-8°C, the volume of the binding solution is CNBr -5-10 times the volume of the agarose affinity chromatography medium, remove the binding solution to obtain an activated CNBr-Sepharose affinity chromatography medium; the activation solution is HCl solution or NaCl-HCl solution, pH 2.0- 4.5; The binding solution is NaCl-NaHCO 3 Solution, pH is 7.0-8.6;

[0071] 2) Weigh the gela...

Embodiment 1

[0087] 1) Weigh 835g of Huiqiong's CNBr-Sepharose affinity chromatography medium in the state of gravity sedimentation and add it to the suction funnel, use a vacuum pump to filter and clean, use 10 times the volume of 4 ℃ pre-cooled activation solution (that is, the activation solution The volume is 10 times that of Huiqiong affinity CNBr-agarose affinity chromatography medium, the activation solution contains 0.001mol / L HCl, the pH of the activation solution is 3.0) Wash for 30min, filter, and remove the filtrate; then use 10 times the volume at 4℃ Pre-cooled binding solution (that is, the volume of binding solution is 10 times the volume of Huiqiong affinity CNBr-agarose affinity chromatography medium, and the binding solution contains 0.1mol / L NaCl and 0.1mol / L NaHCO 3 , The pH of the binding solution is 8.3) Wash, filter, and remove the filtrate; determine the weight of the Huiqiong affinity CNBr-agarose affinity chromatography medium (sedimentation medium) under the state o...

Embodiment 2

[0100] 1) Weigh 835g of Huiqiong's CNBr-Sepharose affinity chromatography medium in the state of gravity sedimentation into the suction filter funnel, use a vacuum pump to filter and clean, use 5 times the volume of 6 ℃ pre-cooled activation solution (that is, the activation solution The volume is 5 times that of Huiqiong affinity CNBr-agarose affinity chromatography medium. The activation solution contains 0.5mol / L NaCl and 0.001mol / L HCl, and the pH is adjusted to 3.0) Wash for 15min, filter, and remove the filtrate; use 5 times more The volume of 6℃ pre-cooled binding solution (that is, the volume of binding solution is 5 times the volume of Huiqiong affinity CNBr-agarose affinity chromatography medium, and the binding solution contains 0.5mol / L NaCl and 0.2mol / L NaHCO 3 , Adjust the pH to 8.3) Wash, measure the weight of the gravity sedimentation medium 1kg, which is the activated Huiqiong affinity CNBr-Sepharose affinity chromatography medium.

[0101] 2) Weigh 10g of gelatin...

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Abstract

The invention relates to preparing of gelatin affinity chromatography media and a method for separation and purification of fibronectin using the gelatin affinity chromatography media. Activation liquid is used for ligand washing activation of CNBr-agarose affinity chromatography media, and then combination liquid is used for washing. A gelatin solution is prepared, the CNBr-agarose affinity chromatography media washed by the combination liquid are added, and temperature control stirring coupling is carried out. Gelatin coupling concentration is detected, combination liquid washing is carried out, and sealing liquid temperature control stirring sealing is carried out. Acid washing liquid and base washing liquid alternating washing is carried out, water washing is then carried out, and the gelatin affinity chromatography media are obtained. The gelatin affinity chromatography media are placed in a chromatographic column. Equilibrium liquid is used for balancing the chromatographic column. Samples flow through the gelatin affinity chromatography media. The preparing method of the gelatin affinity chromatography media has the advantages that purifying efficiency is high, the production cycle is short, and the technology is simple.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a gelatin affinity chromatography medium. Background technique [0002] Fibronectin (abbreviated as FN) is widely distributed on the cell surface and plasma, and plasma fibronectin exists in plasma and body fluids and is soluble. A large number of research results at home and abroad have proved that fibronectin is highly conserved in the evolution process, and fibronectin of various animals has very similar structures, properties and biological functions, so FN from different sources can be used interchangeably. Because elevated plasma fibronectin concentration is common in acute and chronic hepatitis, fatty liver, cirrhosis, obstructive jaundice, cerebrovascular disease, late pregnancy, pregnancy-induced hypertension, pancreatic cancer, lung cancer, cancerous ascites, extensive metastasis of adenocarcinoma, etc. Concentration reduction...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/24B01J20/30C07K1/22B01D15/38
Inventor 易国军汤珊
Owner WUHAN HUIYAN BIOTECH
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