Difference between BCA and Bradford method to determine protein concentration

The two most commonly used methods for protein quantification are the BCA method, Bradford (Coomassie Brilliant Blue) method.

A brief description of the principle and the choice of the experiment is as follows

BCA principle: in alkaline conditions, the protein will reduce Cu2 + to Cu +. Cu + will bind with BCA reagent to form a purple color complex. Absorbance and protein concentration is proportional. The absorbance at 562 nm was measured and compared with the standard curve to calculate the concentration of the protein.

Bradford principle: Coomassie brilliant blue in the acidic solution is brown with the maximum absorption at 465nm. When it binds with the protein, the color turns into blue with the maximum absorption at 595nm. In a certain concentration range, the protein-dye complex at a wavelength of 595 nm is proportional to the protein content, and the amount of protein bound to the protein is determined by measuring the increase in absorption at 595 nm. Protein and Coomassie brilliant blue binding achieve a balance in 2-5min. The reaction is very fast, the complex maintains stable at room temperature in 1 hour.

Difference

BCA method: This method can be compatible with samples up to 5% SDS, 5% Triton X-100, 5% Tween 20, 60, 80. However, this method is affected by chelating agents and slightly higher concentrations of reducing agents. Need to ensure that EDTA is less than 10 mM without EGTA, dithiothreitol (DTT) is less than 1 mM, beta-Mercaptoethanol is less than 0.01 %.

Bradford method: The concentration of ?-Mercaptoethanol in the sample can be up to 1 M and the concentration of dithiothreitol (DTT) can be as high as 5 mM. However, this method is affected by a slightly higher concentration of detergent. Need to ensure that the SDS is less than 0.01%, Triton X-100 is less than 0.05%, Tween 20, 60, 80 is less than 0.015%.