Protein quantification is a commonly used technique in experimental biology to measure the concentration or total amount of protein in a sample. This assay is very important for fields such as biochemistry, molecular biology, medical research, etc.
Figure 1Quantitative proteome analysis.
1. The basic process of protein quantification includes:
1.Sample preparation: Cell extracts, tissue samples, serum, plasma, urine, or other biological samples containing proteins. Make sure the sample is clean and free of impurities. 2.Select assay method: Choose the protein quantification method that suits your experimental needs. 3.Preparation of standard curves: Preparation of a series of standard samples of known concentrations, which are required to determine protein concentrations. 4.Assay sample: Mix the sample with the reagent in the assay method and follow the steps required by the method. Depending on the color, absorbance, or other properties of the reaction product, the results are measured and recorded using a spectrophotometer or other relevant instrument. 5.Calculate the protein concentration: Calculate the protein concentration in the sample based on the standard curve or the formula of the assay method. 2. Common methods for protein quantification include:
1. Bradford protein assay:
1.Principle: This method is based on the color change that occurs when the Comás Brilliant Blue G-250 dye binds to proteins. 2.Advantages: simple operation, high sensitivity. 3.Disadvantages: Interference from detergents and other chemicals. 2. Lowry method:
1.Principle: Based on the fact that copper ions bind to the polypeptide bonds in proteins under alkaline conditions, and further react with the folin-ciocalteu reagent to produce color changes. 2.Advantages: Suitable for lower concentrations of protein. 3.Disadvantages: The steps are more complex than the Bradford method and are susceptible to interference from other substances. 3. BCA method (biscaric acid method):
1.Principle: Similar to the Lowry method, but with BCA reagents, it is more sensitive to copper ions and protein reactions. 2.Advantages: It is suitable for the determination of trace proteins and has good stability. 3.Disadvantages: Longer incubation time required. 4. Ultraviolet absorption spectrometry
1.Principle: Tryptophan and tyrosine in proteins have specific absorption peaks under UV light. 2.Advantages: Fast and does not require the addition of any reagents. 3.Disadvantages: Sensitive to absorption disturbances from other substances, requiring purer samples. 5. Protein quantification based on mass spectrometry technology
1) Label-free quantification
1.Principle: Quantify proteins directly by analyzing the intensity or counting of peptide ions without the use of chemical tags. 2.Advantages: Simplified operation, suitable for a wide range of sample types, and no additional chemical modifications required. 3.Disadvantages: High quality requirements for sample preparation and mass spectrometry operations, and complex data processing and interpretation. 2）itraq/tmt
1.Principle: Proteins in a sample are labeled using special chemical tags. In mass spectrometry, labeling produces specific lysed ions that are used for quantitative analysis. 2.Advantages: Multiple samples can be analyzed at the same time, which increases the throughput of the experiment. 3.Disadvantages: high cost, complex data processing, high requirements for experimental design and execution. 3) Isotope-encoded affinity labeling (ICAT).
1.How it works: Proteins or peptides are labeled using chemical tags containing different isotopes, and then mass spectrometry is used to compare the relative abundance of proteins in different samples. 2.Advantages: Provides accurate comparison of relative amounts of protein and can be used for complex samples. 3.Disadvantages: Cost, complex operation, may not be able to detect untagged proteins. 4）swath-ms
1.Principle: Mass spectrometry data is collected by systematically scanning all predetermined mass windows based on data-independent acquisition. 2.Advantages: Provides high-throughput, high-repeatability quantitative information for complex sample analysis. 3.Disadvantages: Advanced data processing technology is required, and the instrument performance requirements are high. 5) Multiple Reaction Monitoring (MRM).
1.Principle: Specific precursor and product ion pairs are selected for quantification of a specific peptide or protein. 2.Advantages: Highly specific, sensitive, suitable for quantification of low-abundance target proteins. 3.Disadvantages: Only a limited number of target proteins can be quantified at a time, and information about the target protein needs to be known in advance.