What are the most commonly used protein estimation assays?
As promised in our previous post, we will be discussing a little more about some of the most commonly used colorimetric protein assays so let's get started.
While there are a number of colometric assays that can be used to determine the total protein concentration of a sample, the Coomassie (Bradford) and Bicinchroninic Acid (BCA) assays perfectly complement each other and therefore serve as the primary methods for protein quantitation. You can also use several enhanced alternative versions of these assays to satisfy the requirements of your research.
Coomassie (Bradford) Assay
These assays are based on the binding of protein molecules to Coomassie dye under acidic conditions. When the protein molecules bind with Coomassie dye, the color shifts from brown to blue and the change in color density (which is proportional to the protein concentration in the solution) is read at 595 nm. The basic amino acids (arginine, histidine and lysine) contribute to the formation of dye-protein complexes while smaller proteins under 3 kDa do not produce any color changes.
Copper Binding Assays
In the copper ion based protein assays, the protein solution is mixed with an alkaline solution of copper salt. Under alkaline conditions, cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of cupric (Cu2+) to cuprous ions (Cu+). If the alkaline copper is in excess over the amount of peptide bonds, some of the cupric ions (Cu2+) will remain unbound to the peptide bonds and are available for detection. Protein assays based on copper ions can be divided into two groups, assays that detect reduced cuprous ions (Cu+) and that detect unbound cupric (Cu2+) ions.
The cuprous ions are detected either with bicinchoninic acid (BCA) or Folin Reagent (phosphomolybdic/ phosphotungstic acid). Cuprous ions (Cu+) reduction of BCA Reagent produces a purple color that can be read at 562nm. The amount of color produced is proportional to the amount of peptide bonds, i.e. size as well as the amount of protein/peptide.
The presence of tyrosine, tryptophan, cysteine, histidine and asparginine in protein contributes to additional reducing potential and enhances the amount of color produced. Hence, the amount of blue color produced is dependent on the composition of protein molecules. The reaction of cuprous ions (Cu+) with the bicinchoninic acid and color production is similar to that of Folin Reagent.
In the assays based on the detection of unbound cupric ions, the protein solution is mixed with an amount of alkaline copper that is in excess over the amount of peptide bond. The unchelated cupric ions are detected with a color-producing reagent that reacts with cupric ions. The amount of color produced is inversely proportional to the amount of peptide bond.
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