The step of protein isolation and purification is a complex but delicate process aimed at extracting and purifying a single target protein from complex biological samples. These steps usually include several key stages such as sample preparation, preliminary separation, fine separation, purity analysis and purification. Next, we will elaborate on the goals of each step and its specific operation methods:

1. Sample preparation

1. Objective: Process the sample containing the target protein to remove impurities and dissolve and stabilize the protein.

2. Method:

1>Cell disruption: Use mechanical disruption methods (such as high-speed tissue masher, homogenizer, mortar, etc.), osmotic disruption method, repeated freezing and thawing method, ultrasonic method or enzymatic method to disrupt cells or tissues to release proteins .

2>Centrifugation and filtration: remove cell debris, insoluble substances and other impurities to obtain a relatively pure protein solution.

2. Preliminary separation

1. Objective: According to the physical and chemical properties of the protein, select an appropriate separation method to initially isolate the target protein.

2. Method:

1>Ion exchange chromatography: Separate proteins based on their charge properties.

2>Hydrophobic interaction chromatography: Separate proteins based on their hydrophobicity.

3> Affinity chromatography: Separation using the specific binding of proteins to specific ligands.

4>Isoelectric point precipitation method: Use the principle that protein has the lowest solubility at the isoelectric point for separation.

5> Salting out method: Add a large amount of neutral salt to the protein solution to precipitate the protein.

3. Subdivision

1. Objective: To further purify the protein initially separated.

2. Method:

1>Gel filtration chromatography (size exclusion chromatography): Separate proteins based on their molecular size.

2>High performance liquid chromatography (HPLC): Use high pressure to pass the sample through the stationary phase to separate the proteins based on their affinity.

3>Electrophoresis: Use the force of electric field to separate proteins based on their charge and molecular size.

4. Purity analysis

1. Objective: Conduct purity analysis on the separated and purified protein to confirm its purity.

2. Method:

1>SDS-PAGE electrophoresis: Analyze purity by comparing protein mobility.

2>UV-Visible Spectrum: Analyze the purity based on the absorption spectrum of the protein.

3>Mass spectrometry: Analyze purity by measuring the molecular weight of proteins.

5. Refining

1. Objective: Concentrate, crystallize, and other treatments on the purified protein as needed to improve purity and activity.

2. Methods: Including dialysis, ultrafiltration, freeze-drying and other technologies to remove excess salts, buffers or other small molecule impurities while maintaining the natural activity and stability of the protein.

Protein separation and purification is a multi-stage, multi-method process, and it is necessary to select an appropriate separation and purification strategy based on the properties of the target protein and experimental requirements. Each step is critical and requires careful operation and strict control of conditions to ensure that the final target protein is highly pure and highly active.