Quantitative protein measurement, that is, the precise determination of protein concentration in a sample, provides valuable experimental data for scientific researchers. With the continuous advancement of science and technology, methods for quantitative protein measurement are also changing with each passing day, covering many fields such as chemistry, spectroscopy, and biology.
Protein quantitative measurement method
1. Chemical method 1. Principle of Kjeldahl nitrogen determination method: Protein is an organic compound containing nitrogen. It is decomposed into ammonia through digestion, and then combined with sulfuric acid to form ammonium sulfate. Distillation makes the ammonia free. After absorbing it with boric acid, it is converted into ammonia with sulfuric acid or sulfuric acid. Titrate the hydrochloric acid standard solution and calculate the protein content based on the acid consumption. Features: The operation is complex, but the results are relatively accurate. It is one of the classic protein quantification methods. 2. Principle of the biuret method: The protein reacts with the biuret reagent to form a purple-blue complex, and the absorbance is measured at a specific wavelength to calculate the protein content. Features: Easy to operate, suitable for measuring medium concentration protein solutions. 3. Phenol reagent method (Folin-phenol method) Principle: The tyrosine residues in the protein react with the phenol reagent under alkaline conditions to form a blue compound, and the absorbance is measured at a specific wavelength. Features: High sensitivity, but relatively cumbersome operation.
2. Spectral method 1. Principle of ultraviolet absorption method: Tyrosine, tryptophan and phenylalanine in proteins have characteristic maximum absorption at a wavelength of 280nm, which can be used to determine the concentration of protein solutions. Features: Easy to operate, no need to add additional reagents, but may be interfered by impurities such as nucleic acids. 2. Principle of fluorescence method: The protein content is determined by using changes in fluorescence intensity after certain fluorescent dyes bind to proteins. Features: High sensitivity and good selectivity, but may be interfered by other fluorescent substances in the sample.
3. Colorimetric method 1. Principle of Coomassie Brilliant Blue method: The color of Coomassie Brilliant Blue dye changes after binding to protein, and the protein content can be estimated by measuring the color change. Features: Easy to operate, high sensitivity, suitable for many types of proteins. 2. Principle of the BCA method (bicinchoninic acid method): Under alkaline conditions, divalent copper ions are reduced to monovalent copper ions by proteins. Monovalent copper ions react with BCA to form a purple reaction complex, which is calculated by measuring the absorbance. Protein content. Features: High sensitivity, wide detection range, suitable for many types of proteins, and less affected by chemical substances such as detergents.
4. Biological methods 1. Principle of the low Richter method and its derivatives: Use the growth, division or change of proteins on certain organisms to determine the protein quality by measuring changes in the relationship between proteins and organisms. Features: High sensitivity and good repeatability, but the operation is relatively complex.
5. Other methods In addition, there are other methods such as colloidal gold method, silver staining method, etc. These methods may also be used for quantitative measurement of proteins under certain specific conditions.
6. Notes 1. When selecting a specific protein quantitative measurement method, comprehensive considerations should be made based on the experimental purpose, sample characteristics, experimental conditions and other factors. 2. Different methods may be interfered by different factors, such as nucleic acids, detergents, etc. Therefore, when conducting experiments, attention should be paid to controlling the impact of these factors on experimental results. 3. Before conducting the experiment, you should carefully read the operating steps and precautions of the relevant methods to ensure the accuracy and reliability of the experiment.
There are many methods for protein quantitative measurement, each with its own unique principles and scope of application. From classic chemical methods to modern spectroscopic methods, biological methods, and emerging new technologies, the continuous advancement of these methods has provided researchers with more diverse and precise means to determine the protein content in samples. In practical applications, researchers should select the most suitable method for quantitative protein measurement based on comprehensive considerations such as experimental purpose, sample characteristics, experimental conditions, and cost-effectiveness.
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