Bradford's Assay

By Parth Kumar | 28 August 2024

In any Synthetic Biology experiment which involves working with proteins, the first step is quantifying the amount of protein present in the sample - irrespective of which protein it is. Before running an SDS-PAGE, for instance, we must know the concentration of the protein so that we can appropriately decide how much to load. This is done using Bradford's Assay.

When light passes through a sample, the components of the sample absorb some of the light. Bradford's Assay is a colourimetric assay, based on Beer-Lambert's Law - which says that the absorbance (amount of light absorbed as a fraction of the total light incident) of a particular solution is directly proportional to the concentration of the solution.

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Bradford's Assay makes use of the dye CBB G-250 (Coomassie Brilliant Blue). On binding to a protein, the absorbance maximum (wavelength of light absorbed) shifts from 465nm to 595nm. This is because:

  1. CBB donates free electrons to ionisable groups on protein, which disrupts the native state (the active, folded form of the protein) and exposes hydrophobic pockets
  2. Hydrophobic pockets bind to the non polar region of the dye via van der Waals forces. As a consequence, positive amine groups in the protein come tête-à-tête with the negative charge of CBB.
  3. This ionic interaction stabilises the complex

Thus, absorbance is measured at 595nm, and the absorbance is proportional to the amount of bound dye. Using a standard curve of known protein concentrations vs the absorbance, we can find the protein concentration in an unknown sample. This is the utility of Bradford's Assay.

References:

  1. Kielkopf, C. L., Bauer, W., & Urbatsch, I. L. (2020). Bradford Assay for Determining Protein Concentration. Cold Spring Harbor protocols, 2020(4), 102269.

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