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Did you know why a protein's color change reveals its concentration?
Proteins play vital roles in a variety of biological processes, so measuring their concentration is critical for many biomedical research and experiments. However, traditional measurement methods often require specific equipment or complicated steps, making many researchers face challenges when performing protein analysis. To solve these problems, in 1976, Marion M. Bradford invented a simple and rapid technique for measuring protein concentration, called the Bradford protein assay.
The Bradford protein assay uses color change to determine protein concentration, a process that relies primarily on a dye called Coomassie Brilliant Blue G-250.
According to the Bradford method, when a protein solution is combined with Coomassie Gray Blue G-250 dye, the color of the dye changes from its original red color to blue. The key to this change is that the dye will bind non-covalently to certain amino acids in the protein in an acidic environment, thereby changing its absorbance. Specifically, the dye has an absorption peak at 465 nanometers, and when it binds to protein, the absorption peak shifts to 595 nanometers.
This change in absorption can be used to estimate the protein concentration in the sample.
This method not only has the advantages of high sensitivity and simple operation, but also is not easily affected by other chemicals (such as sodium, potassium or certain sugars, etc.). This is very important in many samples containing impurities. However, when the SDS concentration in the sample is too high, it may interfere with the accuracy of the method, causing the protein index to not be correctly guided. When facing this challenge, researchers constantly adjust measurement conditions and explore alternative analysis methods to maintain experimental accuracy.
A key advantage of the Bradford protein assay is that it can be completed in as little as half an hour, saving time and reducing experimental costs. The standard procedure is extremely simple. Just mix the Bradford reagent with the sample, and after a short wait, the photometer measurement can be taken directly. In addition, this method is applicable to almost all types of proteins, especially when it is necessary to quantify trace amounts of proteins. It is extremely sensitive and can accurately measure samples below 20 μg.
The sensitivity and simplicity of the Bradford protein assay allow researchers to use it flexibly to analyze a variety of proteins.
As scientific research advances, the Bradford protein assay also faces some challenges. For example, the assay range is relatively short, typically measuring between 0 μg/mL and 2000 μg/mL, which forces researchers to dilute when analyzing samples, which can lead to measurement errors. In addition, the measurement results of the Bradford method can be biased for certain proteins, especially in samples with higher collagen content, which makes improvement of the method a focus of future development.
Another noteworthy improvement is that adding a small amount of sodium dodecyl sulfate (SDS) during the assay will significantly improve the accuracy of detection of key proteins such as collagen. This improvement not only improves the detection sensitivity of collagen-like proteins, but also reduces the absorption of non-collagen proteins.
As research continues in this area, the Bradford protein assay is becoming more widely used. Not only are they widely used in biology and biomedicine, they further advance the understanding of proteins and their functions. Such technology can not only accelerate the process of protein research, but also provide important basic data for the development of new drug resistance. What clues can the color changes of proteins provide us with that were once unexpected?
