Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Quantitative Protein Determination
Protein Determination Using Coomassie Brilliant Blue

8.18.4.1. Bradford method [38].

Reagent — dye solution. Dissolve 100 mg of Coomassie Brilliant Blue G-250 (Sigma) in 50 mL of 95% ethanol. Add 100 mL of phosphoric acid (85%, w/v) to the solution and bring the volume to 1 L with distilled Water. Filter the solution. Store at 20 °C for 2 weeks. The reagent is commercially available from Bio-Rad and Pierce.

Procedure. Place the protein solution (10—100 µg) into a test tube (12×100 mm) and dilute to 0.1 mL with 0.15 M NaCl solution. Add 5 mL of the dye solution and mix using a vortex mixer. After 2 min, start recording the absorption curve (at 595 nm) against a reference solution (0.1 mL of the appropriate buffer + X mL of the dye solution); continue measurements for ~1 h.

Samples containing 1—10 µg in 0.1 mL of buffer can also be used; in this case, add 1 mL of the dye solution. Measurements are carried out in a 1 mL cuvette.

Cuvettes should be rinsed with an appropriate detergent, water, and acetone to remove the protein-dye complex that forms a film on the Glass surface, or soaked overnight in 0.1 M HCl.

Note. The described method is ~4 times more sensitive than the Lowry Method. The color is stable (±4%) for 1 h. Catecholamines (as well as sodium, potassium, and magnesium chlorides, ammonium sulfate, and ethanol) do not interfere with the assay, which made it suitable for analyzing adrenal protein fractions [306]. Buffer components with alkaline properties, Tris, acetic acid, 2-mercaptoethanol, sucrose, glycerol, and trace amounts of detergents have a minor effect on the results; this Interference can be compensated for by adding the appropriate components to the reference solution. For more details on compounds that interfere with accurate protein determination, see [350].

The advantages of the Bradford method are the speed of analysis and reproducibility of results; however, plots of absorbance at 595 nm versus protein amount are not always linear, and the slope of the curve varies significantly for different Proteins [289, 377]. Therefore, a standard curve generated for a reference protein may not be applicable to an unknown protein without appropriate corrections. The most reliable results [303] are obtained by measuring protein absorbance at 280 nm (at protein concentrations >50 µg/mL) or 205 nm (at protein concentrations <50 µg/mL).

A thorough investigation of the Bradford method [350] revealed that the extinction coefficient of the protein-dye complex remains constant at a protein concentration of 0.8—10 µg/mL; therefore, it is recommended to choose the volume of the dye solution (0.5—5.0 mL) such that the final protein concentration falls within this range. This ensures compliance with the Beer-Lambert law (linearity) and allows for accurate protein quantification within the 0.5—50 µg range.

Proteins at concentrations below 2 µg/mL were determined using Coomassie R (Sigma). The protein was precipitated onto a glass-fiber disc, washed with trichloroacetic acid, treated with the Coomassie reagent, and the resulting colored complex was dissolved in a methanolic NaOH solution for colorimetric determination at 590 nm [249]. The Use of alkali, recommended for releasing the precipitated protein from the support, improves subsequent colorimetric or radiometric measurements [101].

The Bradford method can be applied to proteins dissolved in Electrophoresis buffer containing SDS, mercaptoethanol, and Tris. Under these conditions, BSA exhibits a linear response (in the range of 10—90 µg of protein) similar to that obtained with the Lowry method; when applied to the analysis of Cell homogenates, both Methods yielded reproducible curves with different slopes [323].

8.18.4.2. Micro-assay variant of the Bradford method [46]. Aliquots of protein samples (100 µL each, e.g., HPLC eluate fractions) or standard protein solutions are placed into the wells of a microtiter plate, mixed with 200 µL of dye solution, and diluted with water (1:5). Shake vigorously and, after 10 min, measure the absorbance at 595 nm (Titertek Multiskan spectrophotometer, Flow Labs. Inc. filters).