Protein extraction method

Protein extraction is a commonly used technique in biochemistry and molecular biology and is widely used to efficiently isolate and purify target proteins from diverse biological samples. Given the unique properties of proteins and the specific needs of their experiments, scientists have developed a variety of sophisticated and efficient extraction strategies. Some common protein extraction methods are introduced below:

1. Aqueous solution extraction method

1. Principle: Aqueous solutions of dilute salt and buffer systems have good stability and high solubility for proteins, and are the most commonly used solvents for protein extraction.

2. Steps:

1>Choose an appropriate solvent (such as physiological saline, phosphate buffer, etc.), usually the dosage is 1 to 5 times the volume of the raw material.

Stir evenly to dissolve the protein.

2>The extraction temperature depends on the nature of the active ingredients. Generally, low temperature (below 5°C) operation is used to prevent protein denaturation.

Proteolytic enzyme inhibitors (such as diisopropylfluorophosphate, iodoacetic acid, etc.) can be added to avoid protein degradation.

2. Organic solvent extraction method

1. Principle: Some proteins that are strongly bound to lipids or have more non-polar side chains in their molecules are insoluble in aqueous solutions, but are soluble in organic solvents such as ethanol, acetone, butanol, etc.

2. Steps:

1>Choose a suitable organic solvent as the extraction solution.

2>Operate at low temperature to prevent protein denaturation.

3> Promote protein dissolution through stirring, shaking, etc.

4>Centrifuge to separate the proteins in the extract.

3. Salting out method

1. Principle: The solubility of protein in aqueous solution is affected by salt concentration. Neutral salt can reduce the solubility of protein in water and cause it to precipitate.

2. Steps:

1>Add saturated ammonium sulfate, sodium sulfate and other neutral salt solutions to the protein solution.

2>As the salt concentration increases, the protein gradually precipitates.

3>Centrifuge to separate the precipitated protein.

4. Ultracentrifugation

1. Principle: Use the different sedimentation speeds of different particles in gradient liquid to achieve separation and purification of proteins.

2. Steps:

1>Prepare density gradient solution (such as sucrose, glycerol, etc.).

2>Place the protein solution above the gradient solution.

3>Carry out ultracentrifugation to distribute the protein according to density gradient.

4>Collect proteins from different density layers.

5. Gel chromatography

1. Principle: Use the molecular sieve function of gel to separate protein molecules according to their size.

2. Steps:

1> Load the gel into the chromatography column and equilibrate it with appropriate solution.

2>Add the protein solution to the chromatography column and let it diffuse in the gel.

3>Protein molecules are eluted in order of size, and the eluates at different time periods are collected.

6. Affinity chromatography

1. Principle: Use the specific affinity between biological macromolecules for separation.

2. Steps:

1> Immobilize ligands with specific affinity (such as antibodies, enzyme inhibitors, etc.) on the solid phase matrix.

2> Pass the protein solution through the chromatography column to bind the target protein to the ligand.

3> Wash to remove unbound proteins.

4>Change the elution conditions (such as pH value, ionic strength, etc.) to dissociate the target protein from the ligand and collect it.

7. Precautions

1. Maintain low temperature during the extraction process to prevent protein denaturation.

2. Select appropriate solvents and conditions to ensure protein stability and solubility.

3. The extracted protein should be purified and identified to confirm its purity and activity.

The above are several common protein extraction methods. The specific method to choose depends on the nature of the protein, the purpose of the experiment, and the experimental conditions. In actual operation, adjustments and optimization may need to be made based on specific circumstances.