Bradford protein assay
[1] It is a quick and accurate[2] spectroscopic analytical procedure used to measure the concentration of protein in a solution.
These pockets in the protein's tertiary structure bind non-covalently to the non-polar region of the dye via the first bond interaction (van der Waals forces) which position the positive amine groups in proximity with the negative charge of the dye.
[4] The cationic (unbound) form is green / red and has an absorption spectrum maximum historically held to be at 465 nm.
Additionally, the absorption maxima at 280 nm requires that proteins contain aromatic amino acids such as tyrosine (Y), phenylalanine (F) and/or tryptophan (W).
If nucleic acids are present in the sample, they would also absorb light at 280 nm, skewing the results further.
By using the Bradford protein assay, one can avoid all of these complications by simply mixing the protein samples with the Coomassie brilliant blue G-250 dye (Bradford reagent) and measuring their absorbances at 595 nm, which is in the visible range[8] and may be accurately measured by the use of a mobile smartphone camera.
[2] Additionally, protein binding triggers a metachromatic reaction, evidenced by the emergence of a species that absorbs light around 595 nm, indicative of the unprotonated form[10] This dye creates strong noncovalent bonds with the proteins, via electrostatic interactions with the amino and carboxyl groups, as well as Van Der Waals interactions.
This process is more beneficial since it is less pricey than other methods, easy to use, and has high sensitivity of the dye for protein.
[11] After 5 minutes of incubation, the absorbance can be read at 595 nm using a spectrophotometer or a mobile smartphone camera (RGBradford method).
[9] The Bradford assay is linear over a short range, typically from 0 μg/mL to 2000 μg/mL, often making dilutions of a sample necessary before analysis.
Basic conditions and detergents, such as SDS, can interfere with the dye's ability to bind to the protein through its side chains.
When more than one solution is tested, it is important to make sure every sample is incubated for the same amount of time for accurate comparison.
This is a disadvantage because the preference of the dye to bind to these amino acids can result in a varied response of the assay between different proteins.
One notable modification involves incorporating small amounts, approximately .0035%, of sodium dodecyl sulfate (SDS).
This inclusion of SDS has been shown to result in a fourfold increase in color response for three key collagen proteins—Collagen types I, III, and IV—while simultaneously decreasing the absorbance of non-collagen proteins.
[19] This simple modification in the preparation of the reagent resulted in Bradford Assays to produce similar response curves for both collagen and non-collagen proteins, expanding the use of Bradford Assays in samples containing high collagen proteins.
In summary, in order to find a standard curve, one must use varying concentrations of BSA (Bovine Serum Albumin)[2] in order to create a standard curve with concentration plotted on the x-axis and absorbance plotted on the y-axis.
[24] The equation displayed on the chart gives a means for calculating the absorbance and therefore concentration of the unknown samples.
In a large scale, one must compute the extinction coefficient using the Beer-Lambert Law A=εLC in which A is the measured absorbance, ε is the slope of the standard curve, L is the length of the cuvette, and C is the concentration being determined.
