(1) Pretreatment of materials and cell disruption
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When separating and purifying a certain protein, it is first necessary to release the protein from the tissue or cells and maintain the original natural state without losing activity. Therefore, appropriate methods should be used to break up tissues and cells. Common methods for breaking tissue cells are:
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Mechanical crushing method
This method uses the shearing action of mechanical force to break the cells. Commonly used equipment, high-speed tissue masher, homogenizer, mortar and so on.
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2. Osmosis breaking method
This method causes the cells to swell and break under hypotonic conditions.
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3. Repeated freezing and thawing
After the biological tissue is frozen, the intracellular fluid freezes and the cells swell. This method is simple and convenient, but it should be noted that proteins that are sensitive to temperature changes should not be used.
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4. Ultrasonic method
The ultrasonic oscillator is used to break the cells by uneven tension on the cell membrane.
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5. Enzymatic method
For example, lysozyme is used to destroy microbial cells and the like.
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(2) Extraction of protein
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The protein is usually extracted by selecting an appropriate buffer solvent. The pH, ionic strength, composition and the like of the buffer used for extraction should be selected depending on the nature of the protein to be prepared. For example, in the extraction of membrane proteins, a surfactant (sodium dodecyl sulfate, triton X-100, etc.) is generally added to the extraction buffer to destroy the membrane structure, which facilitates separation of the protein from the membrane. During the extraction process, attention should be paid to the temperature to avoid vigorous agitation, etc., to prevent denaturation of the protein.
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(3) Obtaining crude protein products
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Use the appropriate method to separate the desired protein from other miscellaneous proteins. A more convenient and effective method is the separation based on the difference in protein solubility. There are several methods commonly used:
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Isoelectric precipitation method
Different proteins have different isoelectric points and can be separated from each other by isoelectric precipitation.
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2. Salting out
The salt saturation required for salting out different proteins is different, so the target protein can be precipitated by adjusting the salt concentration. The protein precipitated by salting out retains its natural properties and can be dissolved again without denaturation.
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3. Organic solvent precipitation method
Neutral organic solvents such as ethanol and acetone have lower dielectric constants than water. It can reduce the solubility of most globular proteins in aqueous solution and precipitate out of solution, so it can be used to precipitate proteins. In addition, the organic solvent destroys the hydration layer on the surface of the protein, causing the protein molecules to become unstable and precipitate. Since the organic solvent denatures the protein, when using this method, care should be taken to operate at a low temperature to select a suitable organic solvent concentration.
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(4) Further separation and purification of the sample
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The protein obtained by the isoelectric precipitation method and the salting out method generally contains other protein impurities, which must be further separated and purified to obtain a sample having a certain purity. Commonly used purification methods are: gel filtration chromatography, ion exchange cellulose chromatography, affinity chromatography and the like. Sometimes these methods are used in combination to obtain higher purity protein samples.
Shanghai Jinpeng Analysis Instrument Co., Ltd. Â Â / Â
Telephone 61425398 36162366 Â
400 free consultation phone Â
Mobile phone 18201872685 13764356938
Enterprise QQ, 2880150292, 2880150293, 2880150290, 2880150297
fax
Address: No. 15, Lane 1398, Zhenchen Road, Shanghai, China Postcode: 200444 Â
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When separating and purifying a certain protein, it is first necessary to release the protein from the tissue or cells and maintain the original natural state without losing activity. Therefore, appropriate methods should be used to break up tissues and cells. Common methods for breaking tissue cells are:
Â
Mechanical crushing method
This method uses the shearing action of mechanical force to break the cells. Commonly used equipment, high-speed tissue masher, homogenizer, mortar and so on.
Â
2. Osmosis breaking method
This method causes the cells to swell and break under hypotonic conditions.
Â
3. Repeated freezing and thawing
After the biological tissue is frozen, the intracellular fluid freezes and the cells swell. This method is simple and convenient, but it should be noted that proteins that are sensitive to temperature changes should not be used.
Â
4. Ultrasonic method
The ultrasonic oscillator is used to break the cells by uneven tension on the cell membrane.
Â
5. Enzymatic method
For example, lysozyme is used to destroy microbial cells and the like.
Â
(2) Extraction of protein
Â
The protein is usually extracted by selecting an appropriate buffer solvent. The pH, ionic strength, composition and the like of the buffer used for extraction should be selected depending on the nature of the protein to be prepared. For example, in the extraction of membrane proteins, a surfactant (sodium dodecyl sulfate, triton X-100, etc.) is generally added to the extraction buffer to destroy the membrane structure, which facilitates separation of the protein from the membrane. During the extraction process, attention should be paid to the temperature to avoid vigorous agitation, etc., to prevent denaturation of the protein.
Â
(3) Obtaining crude protein products
Â
Use the appropriate method to separate the desired protein from other miscellaneous proteins. A more convenient and effective method is the separation based on the difference in protein solubility. There are several methods commonly used:
Â
Isoelectric precipitation method
Different proteins have different isoelectric points and can be separated from each other by isoelectric precipitation.
Â
2. Salting out
The salt saturation required for salting out different proteins is different, so the target protein can be precipitated by adjusting the salt concentration. The protein precipitated by salting out retains its natural properties and can be dissolved again without denaturation.
Â
3. Organic solvent precipitation method
Neutral organic solvents such as ethanol and acetone have lower dielectric constants than water. It can reduce the solubility of most globular proteins in aqueous solution and precipitate out of solution, so it can be used to precipitate proteins. In addition, the organic solvent destroys the hydration layer on the surface of the protein, causing the protein molecules to become unstable and precipitate. Since the organic solvent denatures the protein, when using this method, care should be taken to operate at a low temperature to select a suitable organic solvent concentration.
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(4) Further separation and purification of the sample
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The protein obtained by the isoelectric precipitation method and the salting out method generally contains other protein impurities, which must be further separated and purified to obtain a sample having a certain purity. Commonly used purification methods are: gel filtration chromatography, ion exchange cellulose chromatography, affinity chromatography and the like. Sometimes these methods are used in combination to obtain higher purity protein samples.
Shanghai Jinpeng Analysis Instrument Co., Ltd. Â Â / Â
Telephone 61425398 36162366 Â
400 free consultation phone Â
Mobile phone 18201872685 13764356938
Enterprise QQ, 2880150292, 2880150293, 2880150290, 2880150297
fax
Address: No. 15, Lane 1398, Zhenchen Road, Shanghai, China Postcode: 200444 Â
Freeze-dried powder is a sterile powder injection obtained by freezing the liquid medicine into a solid state in a sterile environment, and subliming and drying the water in a vacuum. Freeze-dried powder is composed of a bottle of high-purity and high-active biological protein freeze-dried powder and a high-purity liquid essence. When using, it needs to be connected with a patented vacuum to reconstitute the freeze-dried powder and the essence to activate the biological protein activity.
Freeze Dried Fruit Powder,Strawberry Powder,Freeze Dried Powder,Raspberry Powder
YT(Xi'an) Biochem Co., Ltd. , https://www.ytwholefood.com