Protein purification methods

Protein purification is a core technology in the field of biochemistry and molecular biology. Its purpose is to accurately extract high-purity target proteins from complex biological mixtures. This process not only requires superb technology, but also requires careful selection of the most suitable purification method based on the unique properties of the target protein, the specific goals of purification, and careful consideration of the experimental environment. This article will introduce several protein purification methods suitable for different situations:

1. Solution separation method

1>Principle: Use the difference in solubility of proteins in different solvents for separation.

2>Commonly used solvents: salt solution, organic solvents, etc.

3>Features: Basic and simple, but usually used as a preliminary separation step.

2. Ion exchange chromatography

1>Principle: Use the charge interaction between proteins and ion exchange resin for separation. Immobilized ions on the resin selectively adsorb and release proteins.

2>Type: cation exchange and anion exchange.

3> Features: The purification process has the characteristics of reversibility, strong controllability and sample concentration. It is often used in purification experiments and is often used in combination with other methods.

3. Gel filtration chromatography (also called exclusion chromatography or molecular sieve)

1>Principle: Separate proteins based on their molecular size. Proteins of different sizes are separated due to differences in their diffusion capabilities when passing through a gel chromatography column.

2> Features: Mild operating conditions, no need for organic solvents, and good separation effect on polymer substances. But it is not suitable for purifying proteins with high charge density.

4. Affinity Chromatography

1>Principle: Purification using the specific interaction between proteins and affinity matrix. For example, specific antibodies are used to bind to the target protein and then separated and purified through an antibody column.

2>Features: high selectivity, high purity, fast, concentrated, often used for preliminary purification of recombinant proteins.

5. Electrophoresis purification

1>Principle: Separation method using electric field force and protein charge.

2>Type: including gel electrophoresis (such as polyacrylamide gel electrophoresis and SDS-PAGE) and isoelectric focusing, etc.

3>Features: Separate according to the size, charge and shape of proteins, suitable for protein separation under specific conditions.

6. Liquid Chromatography Purification

1>Principle: Separation is based on the distribution coefficient of proteins in the chromatographic column.

2>Type: including high performance liquid chromatography (HPLC), size exclusion chromatography, reverse phase chromatography, etc.

3> Features: High resolution, high sensitivity, suitable for protein purification in complex samples.

7. Small molecule binding and purification

1>Principle: Small molecules (such as enzyme substrates, inhibitors or ligands) that bind to the target protein are used to purify the target protein.

2> Features: Can selectively bind and purify specific proteins.

8. Other methods

1>Ammonium sulfate precipitation method: often used in the rough separation stage to separate the target protein from other cellular components such as RNA, DNA, etc.

2>Hydrophobic interaction chromatography: Separation using the hydrophobic interaction between proteins and chromatography media, suitable for target proteins with hydrophobic properties.

Summarize

Protein purification is undoubtedly a complex but crucial task. It often requires the clever connection of multiple purification technologies in a layer-by-layer manner to gradually extract higher-purity target proteins from complex mixtures. . This process not only requires in-depth insight into the unique properties and purification needs of the target protein, but also requires comprehensive and careful consideration based on the actual conditions of the experimental conditions. What's more worth mentioning is that with the rapid development of science and technology, new protein purification methods are springing up like mushrooms after a rain, providing scientific researchers with more diverse and efficient choices, and greatly promoting the progress in the fields of biochemistry and molecular biology. Progress and development.