Aim:
To determine the total protein concentration in a given sample
Introduction:
As discussed in the previous lecture, the concentration of proteins can be estimated using various methods. We have discussed the concentration estimation of a pure protein using ultraviolet absorption in the previous lecture. For estimating the total protein in a complex protein mixture, one can use dyes that exhibit changes in their spectral properties on binding to the proteins. Bradford is one such dye-based assay for protein concentration estimation.
The principle underlying Bradford assay is the binding of the Coomassie Blue G250 dye (Figure 5.1) to proteins.
Figure 5.1 Structure of Coomassie Blue G250 |
Free Coomassie Blue G250 can exist in four different ionization states with pKα1, pKα2, and pKα3 of 1.15, 1.82, and 12.4. At pH 0, both the sulfate groups are negatively charged and all three nitrogens are positively charged giving the dye +1 net charge (the red form of the dye). Around pH 1.5, the neutral green form of the dye predominates. At neutral pH, the dye has a net charge of +1 (the blue form of the dye). The red, green, and blue forms of the dye absorb visible radiation with absorption maxima at 470, 650, and 590 nm, respectively. It is the anionic form of the dye that binds to the protein. Binding of the blue form of Coomassie Blue G250 with proteins causes red-shift in its absorption spectrum; the absorption maximum shifts from 590 to 620 nm. It, therefore, looks sensible to record the absorption at 620 nm. The absorbance, however, is recorded at 595 nm to avoid any contribution from the green form of the dye. The dye binds more readily to the cationic residues, lysine and arginine. This implies that the response of the assay would depend on the amino acid composition of protein, the major drawback of the assay. The original assay developed by Bradford shows such variation between different proteins. Several modifications have been introduced into the assay to overcome this drawback; the modified assays, however, are more susceptible to interference by other chemicals than the original assay. The original Bradford assay, therefore, remains the most convenient and widely used method.
In this experiment, we shall be using the standard Bradford assay which is suitable for measuring the protein amount ranging from 10 – 10 μg. A microassay suitable for the protein ranging from 1–10 μg is also briefly discussed.