Protein extraction steps

Protein extraction steps vary depending on the variety of sample types, the unique properties of the protein, and the specific goals of the experiment. The following is a general overview of the protein extraction steps, but in actual practice it will need to be adjusted according to the specific situation:

1. Preparation stage

1. Determine the extraction target and materials: clarify the type of protein to be extracted, and select appropriate biological samples (such as cells, tissues, body fluids, etc.).

2. Prepare extraction solutions and reagents: Prepare appropriate extraction solutions, buffers, protease inhibitors, etc. according to the properties of the protein and experimental needs.

2. Sample processing

1. Sample collection: For cell samples, they can be collected by centrifugation, filtration and other methods; for tissue samples, cutting, grinding and other processing are required.

2. Cleaning and impurity removal: Use pre-cooled physiological saline or PBS and other buffers to clean the samples to remove blood, culture medium and other impurities.

3. Broken cells or tissues

1. Mechanical disruption: Use homogenizer, mortar, high-speed tissue masher and other equipment to disrupt cells or tissues.

2. Physical disruption: such as ultrasonic disruption, freeze-thaw disruption, etc., which break cells or tissues through physical force.

3. Chemical disruption: Use enzymatic hydrolysis, osmotic pressure changes and other methods to disrupt cells or tissues.

4. Protein extraction

1. Add extraction solution: Add an appropriate amount of extraction solution to the broken cell or tissue suspension, usually including buffer, salt solution, detergent and other ingredients.

2. Stirring and incubation: Gently stir the suspension to fully dissolve the protein in the extraction solution, and incubate as needed.

3. Centrifugal separation: Centrifuge the suspension to remove insoluble matter, cell debris and other impurities, and collect the supernatant to become the crude protein solution.

5. Purification and Identification

1. Purification: According to needs, salting out, gel chromatography, affinity chromatography and other methods can be used to purify the crude protein.

2. Identification: Identify the purified protein through electrophoresis, chromatography, mass spectrometry and other methods to confirm its purity, molecular weight, structure and other characteristics.

6. Precautions

1. Keep low temperature: Keep the operation at low temperature as much as possible during the entire extraction process to prevent protein denaturation.

2. Avoid contamination: Pay attention to the cleanliness and aseptic operation of experimental equipment to avoid contamination from external substances.

3. Select appropriate solvents and conditions: Select appropriate solvents and conditions for extraction and purification based on the properties of the protein and experimental needs.

Protein extraction is undoubtedly an intricate and precise process, and every step of the operation needs to be precisely controlled to cope with the ever-changing characteristics of different samples and proteins. In addition, with the continuous development of science and technology, new protein extraction methods and technologies are constantly emerging, providing more choices and possibilities for protein research.