Protein sample preparation is a crucial step in the experiment when conducting protein studies. It directly affects the accuracy and reliability of experimental results, so it is particularly important to understand the principles of protein sample preparation. Protein sample preparation not only requires precise operations, but also requires selecting appropriate treatment methods based on the research goals to ensure that the sample is not contaminated and degraded during extraction, purification, storage, etc.
1. Consistency and prevent degradation 1. Maintain consistency: During the entire experiment, the consistency of sample processing should be maintained as much as possible to avoid introducing artificial errors. Different samples were processed using the same methods and conditions, including the control and experimental groups. 2. Prevent degradation: Protein samples should be processed immediately or stored in refrigerated after collection. When freezing samples, liquid nitrogen or a -80°C freezer should be used to prevent protein degradation. When preparing samples, try to keep the sample at low temperature to slow down the rate of protein degradation. The cooling device can be operated in an ice box or used. Avoid prolonged centrifugation, exposure to high temperatures, pH changes or the presence of enzyme activity to reduce protein degradation.
2. Solubility and impurity removal 1. Improve solubility: Choose an appropriate buffer to dissolve proteins, and the pH value should meet the optimal solubility conditions for the protein. Usually, buffers such as Tris-HCl, PBS, or HEPES are used to ensure that all protein samples to be analyzed are in a dissolved state and are stable and repeatable. 2. Remove impurities: Try to remove impurities that interfere with downstream applications, including but not limited to nucleic acids, tissue fragments or certain high-abundance irrelevant proteins. These impurities may affect protein concentration determination, electrophoretic mobility, and interaction with other molecules.
3. Select the extraction method according to downstream experiments 1. Select the extraction method: According to the specific requirements of the downstream experiments, select the appropriate protein extraction method. For example, if the phosphorylation status of a protein is required, an extraction method that can maintain the phosphorylation of the protein should be used. 2. Type of protein extracted: According to experimental requirements, it is determined whether to extract denatured proteins or active proteins, and whether to extract total proteins or subcellular structural component proteins. Subcellular structure separation can increase the loading volume of low-abundance proteins, reduce detection difficulty, and can also analyze proteins of a certain cell component.
4. Sample treatment and recording 1. Sample treatment: During the preparation process, you should pay attention to using a clean workbench, experimental equipment and gloves to avoid contamination of the sample. All tools, reagent bottle openings and tubes should be kept clean. 2. Detailed recording: During the entire sample preparation process, detailed information should be recorded, including sample source, processing steps, concentration, etc., so that it can be tracked and analyzed in subsequent experiments.
5. Protein quantification and denaturation 1. Protein quantification: It is very important to determine the concentration of protein samples so that the correct sample quantity is used in subsequent experiments. Protein concentration can be determined using methods such as Bradford, BCA or Lowry. 2. Protein denaturation: Denature of proteins may be required according to experimental needs. Commonly used denaturing agents include SDS, etc., and proteins can be denatured by heating.
Protein sample preparation requires a series of principles to ensure the quality and consistency of the sample, thereby obtaining reliable experimental results. These principles cover all aspects from sample collection to treatment, dissolution, impurity removal, extraction method selection, sample processing and recording, and protein quantification and denaturing.
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