Methods for preparing protein samples

Protein sample preparation is a crucial step in modern biological and biochemical experiments, especially in protein analysis, structural research and functional research. Whether it is mass spectrometry, immunoprecipitation, or protein crystallography, the purity and quality of protein samples will directly affect the accuracy of experimental results. In order to ensure the smooth progress of the research, the preparation method of protein samples must be fine and effective. Opentrons editor will introduce several common protein sample preparation methods and provide you with some practical tips.

Protein sample preparation

1. Protein Extraction Method Protein extraction is the first step in sample preparation. Often, we need to extract proteins from cells or tissues. In this step, it is important to select the appropriate buffer, and common protein extraction buffers include buffers containing high concentrations of salt, buffers containing detergents, and buffers containing protease inhibitors. The specific extraction method to choose should be determined based on the nature, source and experimental needs of the target protein. 1. Cell lysis method: Release cell contents by physical methods (such as ultrasonic breaking, high-pressure homogenization) or chemical methods (such as surfactant rupture of cell membranes) to extract proteins. This method is suitable for most cell types, especially animal cells. 2. Tissue breaking method: For solid tissue, liquid nitrogen grinding method or glass bead grinding method can be used. Liquid nitrogen grinding can effectively destroy cell structure and release proteins in cells, while glass bead grinding is suitable for cleavage of fibrous tissue.

2. Protein purification After protein extraction, purification is usually required to remove other impurities such as lipids, nucleic acids and small molecules. Common protein purification methods include: 1. Centrifugation method: Separate cell debris, nucleic acid and other insoluble matters by high-speed centrifugation to obtain a supernatant containing the target protein. 2. Affinity chromatography: Purification is performed by using a specific ligand or antibody to bind to the target protein. For example, purified by a GST affinity column or a nickel ion affinity column using a GST tag or a His tagged fusion protein. 3. Gel filtration chromatography: Separation is performed according to the molecular size of the protein. Small molecules are eluted slowly, while large molecules are eluted faster. 4. Ion exchange chromatography: Separate proteins with different charges according to the charge characteristics of the protein. The interaction between the ion exchanger and the target protein is adjusted by changing the pH or salt concentration of the buffer, thereby achieving separation.

3. Protein Concentration In most experiments, the obtained protein sample needs to be further concentrated. Commonly used concentration methods include: 1. Dialysis method: exchange small molecular substances in the protein solution with buffer through a semipermeable membrane to achieve the purpose of concentrating protein. Dialysis method is suitable for situations where small molecule impurities need to be removed. 2. Ultrafiltration method: Use an ultrafiltration membrane to separate the moisture and small molecules in the solution by centrifugation or pressure to concentrate proteins. 3. Ethanol precipitation method: The protein is precipitated by adding ethanol or other organic solvents, thereby removing impurities in the solution. This method is simple and economical, but attention is needed to be paid to the effect of solvent on protein stability.

4. Protein preservation. Protein sample preservation is also a crucial step. If the protein sample is not processed properly, it may lead to degradation or inactivation. Common protein preservation methods are: 1. Cryopreservation: Aliquot the purified protein solution and cryopreserve, usually stored below -80°C to prevent protein degradation. 2. Add protective agents: Adding glycerin, dithiothreitol (DTT) and other protective agents to the protein solution can protect the protein from oxidation or conformational changes in the frozen state. 3. Freeze-drying method: Freeze the protein solution and remove moisture by vacuum pumping water, converting the sample into a dry state, suitable for long-term storage.

5. Precautions and Optimization In the process of preparing protein samples, ensuring the stability, purity and activity of the samples is the key to success. The following are some optimization techniques: 1. Process the sample in a timely manner: Protein is very easy to degrade during extraction and purification, so the operating time should be minimized, and protease inhibitors should be added in time and kept low temperatures. 2. Avoid repeated freeze-thawing: Freeze-thawing will cause protein denaturation. It is recommended to aliquot the protein samples and store them. 3. Choose the right buffer: Different proteins may be sensitive to ingredients such as pH, salt concentration or detergent, so when preparing samples, you should select the appropriate buffer according to the characteristics of the target protein.

Protein sample preparation is a technically demanding and complex process, covering multiple links from cell breaking, protein extraction, purification, concentration and preservation. Mastering the appropriate preparation method is crucial to ensuring the reliability and accuracy of experimental data.

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