Molecular Biology. Practical Guide - Velikov V.A. 2013

Determination of Protein Concentration
Bradford Assay

The main Methods for determining protein concentration in solution are: 1) the biuret reaction using an alkaline copper salt solution; 2) the Lowry Method using the Lowry-Folin reagent; 3) measurement of optical density in the UV region of the spectrum at 280 nm (absorption band of aromatic Amino Acids) or 205–220 nm (absorption band of peptide groups); 4) dye binding with Coomassie Brilliant Blue R-250, known as the Bradford assay.

The Bradford assay (Bradford, 1976) and the Lowry method (Lowry, 1951) are two of the most widely known analytical techniques. The paper by Lowry et al. (Lowry et al., J. Biol. Chem., 1951, V. 193, P. 265–275) is the most frequently cited scientific publication in the world. Both colorimetric methods offer comparable reliability, and using both may be necessary for quality control purposes.

Coomassie Brilliant Blue R-250 dye dissolved in phosphoric acid exhibits a red-brown color (anionic form), but upon binding with Arginine and hydrophobic amino acid residues of the protein, a blue color develops (λ_max = 595 nm, cationic form). Thus, the increase in solution absorbance at a wavelength of 595 nm is proportional to the protein concentration in the solution.

The method involves adding the dye to the protein solution and measuring the optical density. The obtained values are compared against a standard calibration curve constructed using a protein of known concentration, most commonly bovine serum albumin (BSA).

Compared to the Lowry method, the Bradford assay is simpler, faster, and more sensitive while maintaining the same level of accuracy. It provides reliable protein concentration measurements within the range of 2 μg/mL to 120 μg/mL.

Materials and Equipment

Spectrophotometer, cuvettes, BSA solution (10 mg/mL), test protein solution, Coomassie R-250 dye, orthophosphoric acid.

Solutions

- Bradford reagent. Per 1 L: dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of ethanol, add 100 mL of phosphoric acid, bring to 1 L with Water, and filter through paper. The reagent is highly sensitive to protein (1–2 µg/mL). All glassware must be impeccably clean, otherwise the solution will turn blue and spoil. A fresh, clean solution has a brown color.

Procedure

1. Adjust the sample volume to 0.5 mL with water.

2. Add 0.5 mL of Bradford reagent.

3. Mix and allow the color to develop (from 5 s up to a maximum of 30 min).

4. Measure A₅₉₅ in a 1 mL cuvette.

5. Calculate the protein concentration using a BSA standard calibration curve.