Language
Country/Area
No result found
The magical chemistry of Bradford's method: How to detect the secret of protein with a drop of dye?
Since its development in 1976 by Marion M. Bradford, the Bradford protein assay has become an indispensable tool in biochemical research. This rapid and accurate spectroscopic method is able to measure the concentration of proteins in solution and is known for its dependence on amino acid composition. The Bradford method is not only simple and easy to perform, but also highly sensitive, which makes it more and more widely used in laboratories.
The Bradford method is based on the measurement of the color change of phthalocyanine dyes and is quantitatively measured by observing the interaction between different proteins and the dyes.
Principle
The Bradford assay is a colorimetric protein measurement method based on the absorbance change of Phthalocyanine Blue G-250 dye. The dye comes in three forms: anionic (blue), neutral (green), and cationic (red). Under acidic conditions, the red dye turns blue and binds to the protein being measured. If there is no protein to bind, the solution will remain brown.
The dye binds to the carboxyl and amino groups of proteins through van der Waals forces and charged interactions. During this process, the red phthalocyanine dye transfers free electrons to the ionizable side chains of the protein, destroying its native state and exposing a hydrophilic pocket, which allows the dye to further enhance its binding through van der Waals forces and ionic interactions. Binding to proteins.
AdvantagesWhen the dye binds to protein, the absorbance shifts from 465 nm to 595 nm, making the absorbance reading at 595 nm an indicator of concentration.
The Bradford method can avoid interference from many other protein detection methods and has a high tolerance to substances such as sodium sulphate (SDS), which makes this method applicable in a variety of environments. Many samples cannot be reliably measured in the 280 nm absorption range, and Bradford's method requires only the addition of dye and then measurement at 595 nm.
This makes its use as simple as mixing the sample with phthalocyanine blue dye in a test tube and then reading the absorbance at a wavelength of 595 nm. The method is able to measure from 1 to 20 μg of protein and is very sensitive. The test process usually takes no more than 30 minutes and can be performed at room temperature.
The simplicity and flexibility of the Bradford assay make it a fast and reliable choice for protein detection in your laboratory.
Disadvantages
Although the Bradford method has several advantages, it has a relatively narrow linear range and requires dilution prior to analysis, which can lead to stacking of errors. In addition, when the sample contains certain basic conditions or high concentrations of detergents, it may interfere with the test results. Additionally, this method may not be accurate enough for detecting certain types of proteins, such as collagen.This means that when performing the Bradford test, scientists need to pay attention to the composition and possible effects of the reagents used to ensure the accuracy of the results.
Future Development Direction
As scientific research progresses, the Bradford protein assay continues to evolve. One notable modification is the introduction of a small amount of SDS, which increases the response of collagen detection by four times. At the same time, this also reduces the absorbance of other non-collagen proteins, making the test results more accurate.
Operation process
The standard operating procedure for the Bradford method is very simple. For example, using raw bovine plasma gamma globulin as a protein standard, the parameters are 200-1500 μg/mL. In the test application, you only need to dilute the standard solution of different concentrations, add phthalocyanine blue dye, let it stand for 5 minutes, read the absorbance at 595 nm, draw a standard curve, and calculate the concentration of unknown protein.
The preparation and use of the standard curve makes the Bradford method efficient and accurate in calculating protein concentration.
The Bradford protein assay has emerged in the scientific research field due to its unique and convenient characteristics. As the demand for environmental and sample analysis increases in the future, the applicability and importance of this method will be further enhanced. In this rapidly changing scientific world, can we still find simpler and more effective methods to reveal the mysteries of life?
