1.2.3 Coomassie Brilliant Blue Assay
Good Sensitivity and Repeatability, Rapid Color Development, Weak Detergent Compatibility
Experimental Procedures
1. Preparation of Concentrated Bradford Staining Solution
Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol, add 100 mL of concentrated phosphoric acid, then make up the volume to 200 mL with distilled water. This staining solution can be stored at 4°C and remains stable for at least 6 months.
2. Preparation of Protein Samples for Standard Curve
Use proteins with properties similar to the test sample as the standard whenever possible. For example, when determining antibodies, purified antibodies can be used as the standard. If the test sample is unknown, antibodies can also be used as the standard protein. Typically, the standard curve is plotted for protein concentrations ranging from 20 μg to 150 μg per 100 μL.
3. Color Development
Dilute the concentrated dye-binding solution with distilled water at a ratio of 1:5. If precipitation occurs, remove it by filtration.
Take an appropriate volume of each sample and add 5 mL of the diluted dye-binding solution; let the mixture react for 5–30 minutes. After the dye binds to the protein, the color will change from red to blue. Measure the absorbance at a wavelength of 595 nm.
Note: The color development reaction must not exceed 30 minutes.
4. Concentration Calculation
Calculate the protein concentration of the unknown sample based on the measured absorbance values at 595 nm (A₅₉₅ₙₘ).
