Bradford Method: what it is and how it works

Bradford's Method is a laboratory process used in several sciences. Let's see how it works.

Proteins are macromolecules made up of amino acids. About 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.

Ribosomes are the organelles in charge of assembling these amino acids, giving rise to chains with variable orders and length, or what we know as proteins. These biomolecules are essential to conceive life, since 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, the importance of proteins in the generation of life is more than clear. Today we come to bring you a very interesting mechanism related to this topic, because we tell you everything about the Bradford methodmethod, devised to quantify the protein concentration of a solution.

What is Bradford's method?

Bradford's method (known as Bradfords protein essay) was described, as its name suggests, by the American scientist Marion McKinley Bradford in 1976. First of all, it should be noted that it is a spectrometric method, a term that encompasses a set of laboratory procedures based on the interaction of electromagnetic radiation with an analyte (the component of interest). (the component of interest to be separated from the matrix).

In addition to this, it should be noted that this method is colorimetric in nature, i.e. it results are obtained on the basis of the colors and their concentration in a given solution.. The key to this terminological conglomerate lies in the dye "Coomassie blue", since in Bradford's method the changes in its absorbance are quantified according to certain parameters. This dye appears blue in its anionic form, green in the neutral form and red in the 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 in color, so it is very easy to detect the presence of these macromolecules in the first instance with this methodology.

The chemical basis of the Bradford method

We enter into slightly more complex terrain, as it is time to describe what happens between these molecules beyond the direct color changes. Upon binding to the protein, Coomassie blue in its cationic, double protonated (red) form forms a very strong noncovalent bond with the macromoleculeThe Coomassie blue in its double protonated (red) cationic form forms a very strong non-covalent bond with the macromolecule by van der waals forces and electrostatic interactions.

During the formation of this chemical complex, the dye donates to the ionizable portions of the protein its free electron (remember that cation = positive charge, loses electrons), which causes the disruption of the normal protein state. This exposes certain substances that can generate the bonds described above, which we will not go into here because of their chemical complexity. In summary, it is only necessary to know the following:

Red dye (cationic/non-protein bound) ≠ Blue dye (anionic/protein bound).

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, spectrophotometer readings are taken 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 not only is it 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. Amazing, isn't it?

Bradford's method procedure

In order to perform this methodology, a spectrophotometer is needed, which is not exactly cheap (about 2,000 euros approximately), so it is not something that can be done at home.. This machine is capable of projecting a beam of monochromatic 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 that molecule.

In addition, it should be noted that the reagent is not just "crude" Coomassie blue. One must dissolve 100 milligrams of the dye in 50 milliliters of a 95% ethanol solution and add 100 milliliters of 85% phosphoric acid. In addition, it is necessary to dilute it to one liter once the dye has been dissolved and to 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 investigator has the reagent and the spectrophotometer, the following steps must be followed:

• Prepare the spectrophotometer and check its correct operation.
• Prepare the protein solution to be analyzed. Ideally, the 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 these are the maximum and minimum values.
• Preparation of standards. We are not going to go into their particularities because of the chemical complication they entail.
• Add 5 milliliters of reagent to the solution and let it incubate for 5 minutes.
• Measure the absorbance of the mixture in the spectrophotometer at 595 nm.

The results will appear on the screen of the spectrophotometer, and should be noted by the professional conducting the investigation. Once the results are obtained, it is necessary to create a graph (spectrophotometer curve), it is necessary to create a graph (calibration curve) that plots 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.

Advantages

Bradford's method is very easy to perform for any person involved in the laboratory field, since every biologist and chemist has faced a spectrophotometer at least once during his years of study. From measuring the amount of chlorophyll in a solution by crushing a leaf (typical) to much more complex things, spectrophotometers are widespread in learning environments.

In addition to their ease, it is worth noting that many proteins in their natural state have an extremely low absorption range at 280 nm.. Not even all proteins reach this value, as 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 is needed, which almost no one has, to be able to treat them.

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, proteins are removed from the absorbance spectra of other Biological molecules, which could contaminate the sample.

Summary

In this small chemistry class, we have dived into one of the simplest and easiest protein quantification methods to perform, provided you have the relevant material. However, we must emphasize that, as everything in this life, it is not perfect and infallible either: it is usually necessary to make multiple dilutions of the sample for its analysis (minimum and maximum values from 0 µg/mL to 2000 µg/mL), which can lead to mistakes during the process.

In addition, the presence of detergents and other compounds in the solution can impede the correct performance of the method. Fortunately, there are other reagents that can be added to the mixture to overcome these problems in many cases.

Bibliographical references:

• Compton, S. J., & Jones, C. G. (1985). Mechanism of dye response and interference in the Bradford protein assay. Analytical biochemistry, 151(2), 369-374.
• Ernst, O., & Zor, T. (2010). Linearization of the Bradford protein assay. Journal of visualized experiments: JoVE, (38).
• Friedenauer, S., & Berlet, H. H. (1989). Sensitivity and variability of the Bradford protein assay in the presence of detergents. Analytical biochemistry, 178(2), 263-268.
• He, F. (2011). Bradford protein assay. Bio-protocol, e45-e45.
• Jones, C. G., Hare, J. D., & Compton, S. J. (1989). Measuring plant protein with the Bradford assay. Journal of chemical ecology, 15(3), 979-992.
• López, J., Imperial, S., Valderrama, R., & Navarro, S. (1993). An improved Bradford protein assay for collagen proteins. Clinica chimica acta, 220(1), 91-100.
• Zor, T., & Selinger, Z. (1996). Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Analytical biochemistry, 236(2), 302-308.