Question

why Bradford method is more accurate than UV method when measure the concentration of protein?

Answer 1

Bradford assay

1.      It is method where a colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues in your protein.

2.      Whenas the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm.

3.      Manily , we measure the absorbance of a series of known concentrations of a standard protein, so that BSA, and create a standard curve. So that the standard curve to calculate the concentration of your protein sample based on its absorbance.

4.      Bradford assay is quick, and the reagent is not affected by the presence of reducing agents, like DTT and mercaptoethanol, that may be in buffer.

Bradford method is more accurate than UV method when measure the concentration of protein

Because in the uv the absorbance at 280 nm and the Bradford assay depends on the sequence of your protein. If your protein doesn’t contain a decent number of arginine and/or aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of your protein concentration.

To end with, this technique depends on comparing the absorbance of your protein to that of a standard protein. So, if your protein doesn’t react to the dye in a similar manner as your standard protein, your concentration can be off.