Protein sample preparation is an important link in biology, chemistry and medical research, and it provides the basis for subsequent analysis and experiments. Whether it is proteomics research, enzyme activity analysis, or antibody screening, the preparation of protein samples plays a crucial role.
1. Sample selection and treatment 1. Sample selection: Select appropriate tissue or cell as the sample according to the experimental purpose. For tissue samples, it is necessary to ensure fresh treatment and remove connective tissue and adipose tissue. If necessary, the blood inside the organ can be washed away by perfusion. For cell samples, different centrifugation and collection methods can be used depending on whether they are suspended or adherent cells. 2. Sample fragmentation: In order to release proteins in cells, the sample needs to be broken. Commonly used methods include grinding, homogenization, ultrasound, enzymatic lysis, etc. Pay attention to keeping the temperature low during the crushing process to reduce protein degradation.
2. Protein extraction 1. Select a lysate: Select the appropriate lysate according to the sample type and subsequent experimental needs. Commonly used lysates include RIPA, NP-40, SDS, etc. They can destroy cell membranes or cell walls and release proteins. 2. Lysis operation: Add the lysate to the sample on ice and perform sufficient homogenization or grinding to ensure complete release of protein. After lysis, the insoluble substances are removed by centrifugation to obtain a protein supernatant.
3. Determination of protein concentration 1. Determination method: Use the BCA method, Bradford method or Lowry method to determine the protein concentration. These methods are based on the principle that the protein reacts with specific reagents to produce color changes. The concentration of protein can be determined through colorimetric method. 2. Concentration adjustment: Adjust the protein concentration according to the measurement results to make the experimental conditions uniform. This usually involves diluting or concentrating a protein sample to an appropriate concentration range.
4. Protein denaturation (if required) 1. Denaturation purpose: In some experiments, it is necessary to denature the protein to change its conformation so that it is easier to isolate and detect. Commonly used denaturing agents include SDS, urea, etc. 2. Denaturation operation: Add the denaturant to the protein sample and heat the protein (such as heating at 95°C for 5 minutes) to denature the protein. Denaturated protein samples can be used directly for subsequent electrophoresis or immunoblotting experiments.
5. Sample storage and recording 1. Sample storage: Aliquot the prepared protein samples and store them under appropriate conditions (such as -80°C refrigerator) to prevent degradation and contamination. 2. Record information: Record the source, processing steps, concentration measurement results and other information of the sample in detail, so as to analyze and track subsequent experiments. It should be noted that during the entire protein sample preparation process, sterile operation specifications should be strictly followed to avoid contamination. At the same time, appropriate lysate and antibodies should be selected according to experimental needs to obtain the best experimental results. In addition, the preparation process can be adjusted and optimized according to specific experimental needs.
The basic process of protein sample preparation includes sample selection and processing, protein extraction, protein concentration determination, protein denaturation (if required), and sample storage and recording. Together, these steps ensure the quality and reliability of protein samples, providing a solid foundation for subsequent experimental analysis.
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