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Preparation of commodity type affinity column and its operation mode

A chromatographic column and affinity technology, applied in the biological field, can solve the problems of long time-consuming and low final yield

Inactive Publication Date: 2005-06-01
BEIJING ZHONGTIANKANGTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Purification of a single protein from tissue extracts or protein-mixed solutions through the differences in various physical and chemical properties between molecules requires a series of very complicated and cumbersome operations, which take a long time and the final yield is very low.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of affinity chromatography column:

[0024] Take 1 package of 5g Sepharose 4B affinity chromatography column packing that has been coupled with HSP-60 antibody and put it into a bottle with a cover, soak it with 50ml 0.15Mol / L PBS (pH7.2) PBS buffer solution (referred to as PBS buffer solution) After washing, the excess solution is aspirated and the resin is ready for affinity reaction.

[0025] Preparation of interstitial fluid:

[0026] The tumor mass was taken out from the rat, the normal tissue was trimmed, and the tumor tissue cut into small pieces was frozen in a -20°C refrigerator for 2 hours, and then ground in a homogenizer. Gradually add 20ml of chilled PBS buffer to the grinder and continue grinding. The grinding solution was centrifuged at 8000rpm in a refrigerated centrifuge for 10 minutes, and then the supernatant was taken for affinity reaction.

[0027] Affinity reaction:

[0028] Transfer the centrifuged grinding supernatant to a bottle ...

Embodiment 2

[0039] Take a Sepherose 4B affinity chromatography column coupled with HSP-60 antibody and wash it with 0.15Mol / L PBS buffer (pH7.2) to equilibrate for sample loading.

[0040] Sample pretreatment:

[0041] The mixed protein mixture containing HSP-60 molecules was dialyzed against PBS buffer overnight at 4°C.

[0042] Affinity reaction:

[0043] The next day, add the dialyzed miscellaneous protein solution to the equilibrated affinity chromatography column, and keep the flow rate at 0.5ml / min. After the sample flows out, wash the affinity column with a large amount of PBS until the UV absorption value of the effluent is OD 280 <0.02.

[0044] Elution affinity:

[0045] Use 0.1Mol / L glycine-HCl (pH2.4) to elute the target protein bound to the resin-coupled antibody, ie HSP-60 protein. Fractions containing the eluate were collected and immediately dialyzed against PBS buffer overnight at 4°C. Among them, the PBS buffer was changed 1 to 2 times.

[0046] Identification of ...

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PUM

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Abstract

The present invention relates to the application of antibody capable of distinguishing heat shock protein (HSP) in preparing commercial affinity resin and affinity chromatographic column, and aims at extracting HSP and heat shock protein-polypeptide complexes formed with the HSP and non-covalent combined polypeptide antigen molecules fast, effectively and specifically from protein mixing liquid.

Description

technical field [0001] The invention belongs to the field of biotechnology technical background [0002] Purifying a single protein from tissue extracts or protein-mixed solutions through the differences in various physical and chemical properties between molecules requires a series of very complicated and cumbersome operations, which take a long time and the final yield is very low. With the development of biochemical technology, a method for separating and purifying biological macromolecules with simple operation and high separation specificity—affinity chromatography—has emerged. Its principle is to use the specific recognition and binding characteristics between antigen-antibody to purify the target protein in a "one-step method". Its specific implementation method is to couple the antibody capable of recognizing and binding the target protein to a solid-phase resin. Commonly used resins include cyanogen bromide-activated agarose gel, such as Sepharo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/08C07K1/22
Inventor 韩苏
Owner BEIJING ZHONGTIANKANGTAI BIOTECH
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