Bradford Protein Concentration

Bradford method, also known as the Coomassie Brilliant Blue method, is a fast and simple method for determining protein concentration. The method is based on the binding of proteins with a dye (Coomassie Brilliant Blue G-250), and protein concentration is determined by colorimetry.

 

Coomassie Brilliant Blue is brownish-red in its free state, with a maximum light absorption around 490nm. In an acidic solution, the basic and aromatic amino acid residues in the protein bind with Coomassie Brilliant Blue G-250 to form a blue complex, which has maximum light absorption at a wavelength of 595nm. The light absorption value of this complex is proportional to the protein concentration, so it can be used for quantitative determination of proteins.

 

Analysis Workflow

1. Reagent Preparation

Dissolve 100mg of Coomassie Brilliant Blue G-250 in 50mL of 95% ethanol, then add 100mL of 85% phosphoric acid, and finally add distilled water to 200mL. (Can be stored at 4℃ and remain stable for at least 6 months)

 

2. Sample Preparation

Use a protein similar in nature to the sample to be tested as a standard. For example, when determining antibodies, purified antibodies can be used as standards. If the sample to be tested is unknown, antibodies can also be used as standard proteins. A standard curve is usually plotted between 20ug-150ug/100μL.

 

3. Sample Determination

Dissolve the sample to be tested in the buffer solution, which should be the same as the buffer solution used to make the standard curve. Add 1mL of Bradford reagent to 100μL of the sample, and mix well with a high-speed stirrer. Measure the absorbance of the mixed solution at 595 nm within 5-30 minutes.

 

4. Protein Concentration Analysis

Import the absorbance data of the standard protein into Excel to make a scatter plot and get the standard curve formula. The concentration of the unknown protein can be obtained by importing the absorbance data of the unknown protein into the standard curve formula.