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Cell membrane protein enrichment and purification method

A purification method and cell membrane technology, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve the problems of low purity of cell membrane protein extraction, membrane organelle contamination, etc., to achieve effective separation, reduce pollution, and high purity Effect

Active Publication Date: 2015-05-20
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

[0006] In order to overcome the shortcomings of low purity of cell membrane protein extraction and serious contamination of membrane organelles, the present invention provides a cell membrane protein enrichment and purification method, which uses functionalized magnetic microspheres to capture glycosylated proteins on the cell surface as bait for enrichment and analysis. Cell membrane protein, so as to realize the separation of cell membrane and organelles and obtain high-purity cell membrane protein

Method used

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  • Cell membrane protein enrichment and purification method
  • Cell membrane protein enrichment and purification method
  • Cell membrane protein enrichment and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Oxidation of glycoproteins on the cell membrane surface

[0023] After digesting human cervical cancer cells in the culture dish with trypsin, add 5mM sodium periodate solution prepared by oxidation buffer (100mM sodium acetate, 150mM sodium chloride, pH5.5) to the cell sample to react with the cells for 1h;

[0024] 2. Capture of Glycoproteins on the Cell Membrane Surface

[0025] After washing the oxidized cell sample with oxidation buffer to remove the remaining oxidant, add hydrazide magnetic microspheres suspended in oxidation buffer to the oxidized cell sample, and shake at room temperature for 16 hours;

[0026] 3. Blocking of aldehyde groups

[0027] The magnetic microspheres capturing the protein samples were washed with 100 mM tris buffer at pH 8, and the magnetic microspheres capturing the cell samples were resuspended in the tris buffer and incubated for 1 h;

[0028] 4. Removal of Soluble Proteins

[0029] Wash the magnetic microspheres with 2M potass...

Embodiment 2

[0045] The human cervical cancer cells in Example 1 were replaced with human liver cancer cells; the cell sample processing steps were the same as in Example 1.

[0046] Identification result

[0047]

Embodiment 3

[0049] The human cervical cancer cells in Example 1 were replaced with human T lymphoma cells; the cell sample processing steps were the same as in Example 1.

[0050] Identification result

[0051]

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Abstract

The invention relates to a cell membrane protein enrichment and purification method. The method utilizes the natural characteristics that cell membrane surface integrated membrane protein runs through a membrane bimolecular layer and galactosylated modification often occurs to the cell membrane surface membrane protein, and cell membrane surface galactosylated modification protein serving as the bait is capture by functional magnetic microspheres so as to realize separation of the cell membrane and organelles and efficient enrichment of cell membrane integrated membrane protein. The method has the advantages of high separation efficiency of cell membrane and organelles, and can achieve efficient analysis and characterization of cell membrane proteins. In addition, with the introduction of functional magnetic microspheres, the method has the advantages of simple operation, rapid separation and low sample loss.

Description

technical field [0001] The invention relates to a cell membrane protein enrichment and purification method. Background technique [0002] The cell membrane is the interface between the internal and external environment of the cell. Cell membrane proteins play an important role in the exchange of substances inside and outside the cell, cell recognition and immune response signal transduction and regulation, and energy transfer. About 70 percent of known drug targets are cell membrane proteins. Therefore, overexpressed cell membrane proteins of diseased cells can be used as protein targets for therapeutic antibodies and small molecule drugs. [0003] However, among the data structures of more than 86,000 proteins that have been successfully analyzed in the PDB database, only 1% to 2% of the results are related to cell membrane proteins. The reason is that cell membrane proteins have the characteristics of low abundance, strong hydrophobicity, and post-translational modificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 张丽华方菲赵群随志刚杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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