Proteins are macromolecules made up of amino acids. Some 500 different amino acids have been described in nature, but curiously, only 20 are the essential ones present in the human body. DNA contains all the information necessary for a protein to be synthesized, since through transcription and translation mechanisms, a triplet of DNA nucleotides is converted into a specific amino acid. Bradford protein assay procedure
Ribosomes are the organelles responsible for assembling these amino acids, giving rise to chains with orders and variable length, or what is the same, what we know as proteins. These biomolecules are essential to conceive life, as they account for approximately 80% of the dry protoplasm in every cell and represent 50% of the weight in all living tissues.
With these data in hand, it is more than clear to us the importance of proteins in the generation of life. Today we come to bring you a very interesting mechanism related to this topic, because we will tell you everything about the Bradford method , designed to quantify the protein concentration of a solution.
What is the Bradford method?
The Bradford method (known as Bradford’s protein essay in English) was described, as its name suggests, by the American scientist Marion Mckinley Bradford, in 1976. First of all, it is necessary to emphasize that it is a spectrometric method, term that encompasses a set of laboratory procedures based on the interaction of electromagnetic radiation with an analyte (the component of interest to be separated from the matrix).
In addition to this, it should be noted that it is a method of a colorimetric nature, that is, it obtains results based on the colors and their concentration in a specific solution . The key to this terminological conglomerate is found in the “Coomassie blue” dye, since the Bradford method quantifies the changes in its absorbance according to certain parameters. This dye appears blue in its anionic form, green in its neutral form, and red in its cationic form.
Under acidic conditions in solution, Coomassie blue turns from red to blue and, in the process, binds to the proteins to be quantified. If there are no proteins in the aqueous medium, the mixture remains brown, so it is very easy to detect the presence of these macromolecules in the first instance with this methodology. Bradford protein assay procedure
The chemical bases of the Bradford method
We are entering a little more complex terrain, as it is time to describe what happens between these molecules beyond direct color changes. When binding with the protein, Coomassie blue in its cationic and double protonated form (red) forms a very strong non-covalent bond with said macromolecule , through van der waals forces and electrostatic interactions.
During the formation of this chemical complex, the dye donates its free electron to the ionizable portions of the protein (remember that cation = positive charge, it loses electrons), which causes the disruption of the normal protein state. This exposes certain substances that may generate the previously described unions, in which we are not going to stop due to their chemical complexity. In summary, you only need to know the following:
Red dye (cationic / not bound to protein) ≠ Blue dye (anionic / bound to protein)
Based on this premise, it should be noted that the red dye has an absorption spectrum of 465 nm, a value that represents the incident electromagnetic radiation that a material absorbs within a range of frequencies . In the anionic blue form (interacting with proteins), a change in absorption occurs at 595 nm. Therefore, in a solution subjected to the Bradford method, readings are made in spectrophotometers at a range of 595 nm.
The increase in absorbance in this spectrum is directly proportional to the number of bonds between the dye and the proteins, so it is not only detected that there are proteins with the color change, but it is also possible to estimate how much protein there is per milliliter of liquid medium. Incredible true? Bradford protein assay procedure
Bradford method procedure
In order to carry out this methodology, a spectrophotometer is necessary, which is not exactly cheap (approximately 2,000 euros), so it is not something that can be run from home . This machine is capable of projecting a monochromatic beam of light through a sample, in order to measure the amount of light that is absorbed by the compounds of interest. Thus, the researcher receives information about the nature of the molecules in the solution in question and, incidentally, is also able to calculate the concentration of said molecule.
Furthermore, it should be noted that the reagent is not just “raw” Coomassie blue. 100 milligrams of the dye should be dissolved in 50 milliliters of a 95% ethanol solution and 100 milliliters of 85% phosphoric acid added. In addition, it is necessary to dilute it to a liter once the dye has dissolved and filter the mixture, to give rise to the definitive reagent used in the method. The color of this solution without proteins present, as we have said, should be brownish .
Once the researcher has the reagent and the spectrophotometer, he must follow the following steps:
- Prepare the spectrophotometer and check its correct operation.
- Prepare the protein solution to be analyzed. Ideally, this sample should contain between 5 and 100 micrograms of protein per 100 microliters of total solution. It is obvious that the exact concentration is not known, but they are the maximum and minimum values.
- Prepare standards. We are not going to go into their peculiarities due to the chemical complication they entail. Bradford protein assay procedure
- Add 5 milliliters of reagent to the solution and let it incubate for 5 minutes.
- Measure the absorbance of the mixture on the spectrophotometer at 595 nm.
The results will appear on the screen of the spectrophotometer, and should be noted by the professional who is conducting the investigation. Once they are obtained, it is necessary to create a graph (calibration curve) that faces two values on their axes: absorbance vs micrograms of protein . From the curve generated with the values, these can be extrapolated to obtain the exact concentration of protein in the solution.
The Bradford method is very easy to perform for anyone related to the laboratory field, since every biologist and chemist has faced a spectrophotometer at least once during their years of study. Whether it is to measure the amount of chlorophyll in a solution from the crushing of a leaf (typical) to much more complex things, spectrophotometers are very widespread in learning fields.
In addition to its ease, it should be noted that many proteins in their natural state have an extremely low absorption range, at 280 nm . Not even all proteins reach this value, because for this they must have specific amino acids (tyrosine, phenylalanine and tryptophan), which are not always present. As this absorbance figure is in the UV range, a special machine that almost no one has is necessary to be able to treat them. Bradford protein assay procedure
Actually, what is done in the Bradford method is to “increase” the absorbance value of the proteins by binding to a dye . In addition to being much easier to read in this state, the proteins move away from the absorbance spectra of other biological molecules, which could contaminate the sample.
In this small chemistry class, we have immersed ourselves in one of the simplest and easiest protein quantification methods to perform, provided the relevant material is available. In any case, we must emphasize that, like everything in life, it is not perfect and infallible either: it is usually necessary to make multiple dilutions of the sample for analysis (minimum and maximum values of 0 µg / mL to 2000 µg / mL), which which can lead to mistakes during the process.
Furthermore, the presence of detergents and other compounds in the solution can prevent the correct development of the method. Fortunately, there are other reagents that can be added to the mix to solve these problems in many cases. Bradford protein assay procedure