The general procedure for isolating and purifying a specific protein typically involves three main stages: sample preparation, initial fractionation, and final purification.
1. **Sample Preparation**: The first step is to release the target protein from its original cellular or tissue environment in a soluble form while preserving its native structure. If this isn’t possible—such as when the protein exists as inclusion bodies—it’s important to ensure that it retains its biological activity. For animal tissues, it's crucial to remove connective and fatty tissues first. Plant materials should be dehusked or deseeded to avoid contamination by tannins. Using low-boiling organic solvents like ether is often recommended for extraction. After that, depending on the material, appropriate methods are used to disrupt the cells. Animal cells can be broken using sonication, homogenization, or electric mashing. Plant cells, which have rigid cell walls made of cellulose, hemicellulose, and pectin, are usually ground with quartz sand or glass powder, or treated with enzymes like cellulase. Bacterial cells are more challenging due to their tough peptidoglycan cell walls. Common methods include sonication, bead milling, high-pressure homogenization, or treatment with lysozyme. Once the cells are disrupted, the protein is extracted using an appropriate buffer, and insoluble debris is removed through centrifugation or filtration.
If the protein is localized within a specific cellular compartment—like the nucleus, ribosomes, or cytoplasm—differential centrifugation can be used to isolate that fraction for further processing. If the protein is membrane-bound, the membrane must be disrupted using detergents or ultrasound before extraction.
2. **Initial Fractionation**: After obtaining the crude protein extract (which may contain nucleic acids, polysaccharides, etc.), various techniques are applied to separate the desired protein from other contaminants. Common methods include salting out, isoelectric precipitation, and organic solvent fractionation. These approaches are efficient, scalable, and effective at removing impurities and concentrating the protein solution. In some cases, where precipitation is not suitable, ultrafiltration, gel filtration, or freeze-drying may be used to reduce the volume.
3. **Final Purification**: After initial fractionation, the sample becomes more concentrated and contains fewer impurities. At this stage, chromatographic techniques such as gel filtration, ion exchange, affinity, and adsorption chromatography are widely used. Electrophoretic methods like zone electrophoresis or isoelectric focusing may also be employed for higher resolution. These steps are usually performed on a smaller scale but offer greater precision.
4. **Crystallization**: Crystallization is the final step in protein purification. Although it doesn’t guarantee 100% purity, it indicates that the protein is present in sufficient quantity and has reached a high level of homogeneity. Crystallization also helps remove minor impurities, and since denatured proteins rarely crystallize, it serves as a strong indicator of the protein’s native state and quality.
**Common Techniques for Protein Separation and Purification**:
- **Based on Molecular Size**:
- Dialysis and ultrafiltration
- Density gradient centrifugation
- Gel filtration
- **Based on Solubility Differences**:
- Isoelectric precipitation and pH adjustment
- Salting-out and salt solutions
- Organic solvent fractionation
- Temperature effects on solubility
- **Based on Charge Differences**:
- Electrophoresis
- Polyacrylamide gel electrophoresis (PAGE)
- Capillary electrophoresis
- Isoelectric focusing
- Ion exchange chromatography
- **Based on Selective Adsorption**:
- Hydroxyapatite chromatography
- Hydrophobic interaction chromatography
- **Based on Specific Biological Affinity**:
- Affinity chromatography (including lectin, immunoaffinity, metal chelate, dye ligand, and covalent chromatography)
Additionally, high-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC) are commonly used for advanced purification. Each method has its own advantages and is selected based on the properties of the target protein and the goals of the purification process.
Â
Folding Desk,Folding Table,Folding Office Table,Folding Laptop Table,Folding Computer Desk
Suzhou Uplift Intelligent Technology Co., Ltd , https://www.upliftec.com