Protein assays are used to measure the concentration of proteins in a sample, which is essential for downstream applications such as enzyme assays, Western blotting, and protein purification. Several protein assays exist, with the Bradford assay, BCA assay, and Lowry assay being the most commonly used. Below is a detailed protocol for the Bradford protein assay, a widely used and rapid method based on the binding of Coomassie Brilliant Blue dye to proteins.
Bradford Protein Assay Protocol
Materials Needed
Bradford reagent: Commercially available as a ready-to-use solution or prepared by dissolving Coomassie Brilliant Blue G-250 in phosphoric acid and ethanol.
Protein standard: Bovine Serum Albumin (BSA) or other known protein for generating a standard curve.
Unknown protein samples.
Distilled water or buffer: Used to dilute the protein samples and standards.
Microplate reader or spectrophotometer: Set to measure absorbance at 595 nm.
Cuvettes or 96-well microplate: For sample measurement.
Pipettes and tips.
Preparation of Bradford Reagent
If not using a commercially available Bradford reagent, it can be prepared by:
Dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of ethanol.
Adding 100 mL of 85% phosphoric acid.
Diluting to 1 liter with distilled water.
Filter the solution and store in an amber bottle at room temperature.
Step-by-Step Procedure
- Prepare Protein Standards
Prepare a series of BSA standards (or another standard protein) by serially diluting a known concentration of BSA (e.g., 1 mg/mL) to generate a range of concentrations, typically between 0–1 mg/mL.
Example standard curve dilutions:
0 µg/µL (blank)
2 µg/µL
4 µg/µL
6 µg/µL
8 µg/µL
10 µg/µL
Dilute the BSA standards with distilled water or the same buffer used in the unknown protein samples.
- Dilute the Unknown Protein Samples
If the concentration of the unknown protein is unknown, make serial dilutions of your sample to ensure that the protein concentration falls within the range of the standard curve.
For high-concentration protein samples, you may need to dilute the sample in water or buffer.
- Add Bradford Reagent
Microplate method:
Pipette 10 µL of each protein standard and unknown sample into separate wells of a 96-well plate.
Add 200 µL of Bradford reagent to each well.
Mix the contents of each well by gently tapping the plate or using a plate shaker.
Cuvette method:
Pipette 20 µL of each protein standard and unknown sample into cuvettes.
Add 1 mL of Bradford reagent to each cuvette and mix thoroughly.
- Incubate the Reaction
Allow the reaction to incubate at room temperature for 5–10 minutes, but no longer than 1 hour.
During this time, the Coomassie dye binds to the protein, shifting its absorption maximum from 465 nm to 595 nm, resulting in a color change from brownish-red to blue.
- Measure Absorbance
Use a microplate reader or spectrophotometer set to 595 nm to measure the absorbance of each well or cuvette.
Measure the absorbance of the blank (0 µg/µL BSA) and subtract this value from all other absorbance values to account for background absorbance.
- Generate a Standard Curve
Plot the absorbance values of the BSA standards against their known concentrations to generate a standard curve.
The plot should show a linear relationship between protein concentration and absorbance within the range of the assay.
Fit a trendline to the standard curve and use the equation of the line (typically y = mx + b, where y is absorbance, x is protein concentration, m is the slope, and b is the intercept) to determine the concentration of protein in the unknown samples.
- Calculate Protein Concentration of Unknown Samples
Use the absorbance values of the unknown samples to interpolate their concentrations from the standard curve.
Apply the equation obtained from the standard curve to calculate the protein concentration of each unknown sample:
Protein concentration (µg/µL) = (Absorbance b) / m
Multiply by the dilution factor if the unknown sample was diluted before performing the assay.
Notes:
Linear range: The linear range of the Bradford assay is typically between 0.1 to 1.0 mg/mL. If the absorbance of an unknown sample is above this range, dilute the sample and repeat the measurement.
Interfering substances: Detergents (e.g., SDS), high concentrations of salts, and certain buffers (e.g., Tris) can interfere with the Bradford assay. If interference is suspected, switch to an alternative assay like the BCA assay.
Optional: BCA Assay Protocol
For proteins in detergents or other problematic solutions, the BCA (Bicinchoninic Acid) Assay may be more suitable. This assay is compatible with detergents and measures protein concentration by producing a purple-colored complex with proteins that absorb at 562 nm.
Troubleshooting
Low absorbance values: If absorbance values are too low, check that the protein concentration is within the standard curve range. If not, concentrate the sample or prepare fresh standards.
High absorbance values: If absorbance values are too high, dilute the protein samples to bring them within the assay’s linear range.
By following these steps, you can accurately determine the concentration of protein in your samples using the Bradford protein assay.