Protein quantification is a crucial step in many biochemical experiments, and the Bradford protein assay is one of the most widely used methods for this purpose.
Principle of Bradford Protein Assay
The Bradford protein assay is based on the principle that the binding of a dye molecule, Coomassie Brilliant Blue, to proteins results in a shift in the dye's absorption spectrum. When the dye binds to protein, its absorption maximum shifts from 465 nm to 595 nm, resulting in a change in color from brown to blue. The amount of protein in a sample is determined by measuring the absorbance of the blue-colored solution at 595 nm and comparing it to a standard curve generated using known concentrations of protein.
Why Perform Bradford Protein Assay?
The Bradford protein assay is widely used because it is simple, rapid, and highly sensitive. It can detect as little as 1 microgram of protein in a sample, making it suitable for use with small amounts of protein, such as those obtained from cell culture or tissue samples. The assay is also compatible with a wide range of sample types, including serum, plasma, cell lysates, and purified protein solutions.
Protocol of Bradford Protein Assay
Materials:
Bradford reagent
Protein sample
Bovine serum albumin (BSA) standard solutions
Spectrophotometer
Cuvettes
Procedure:
- Prepare the Bradford reagent according to the manufacturer's instructions. Briefly, the Bradford reagent can be prepared by dissolving 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol. Then, add 100 mL of 85% phosphoric acid and 850 mL of distilled water. Mix well and store the solution in a brown glass bottle.
- Prepare a series of BSA standard solutions of known concentrations (e.g. 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) by diluting a stock solution of BSA in distilled water.
- Dilute the protein sample to an appropriate concentration using distilled water. The concentration of the protein sample should be within the linear range of the standard curve.
- Add 20 µL of the protein sample or BSA standard to a cuvette containing 980 µL of distilled water. Then, add 1 mL of Bradford reagent to each cuvette and mix well by inverting the cuvette several times.
- Incubate the cuvettes at room temperature for 5 minutes. During this time, the Bradford reagent binds to the protein, resulting in a shift in the dye's absorption spectrum and a change in color from brown to blue.
- Measure the absorbance of the samples and standard at 595 nm using a spectrophotometer. Set the spectrophotometer to zero using a blank cuvette filled with distilled water. Then, measure the absorbance of the standard solutions and the protein sample.
- Calculate the protein concentration of the samples by comparing the absorbance values to the standard curve obtained from the BSA standard solutions. Plot the absorbance values of the standard solutions against their corresponding protein concentrations to create a standard curve. Use the standard curve to determine the protein concentration of the sample based on its absorbance.
- Repeat steps 4 to 7 for each sample and standard to obtain multiple measurements.
- Calculate the mean protein concentration of the samples by averaging the results of the multiple measurements.
Tips for a Successful Bradford Protein Assay:
- Ensure that the Bradford reagent is freshly prepared and stored in a brown glass bottle to prevent photodegradation.
- Use distilled water to prepare all solutions and dilute the protein sample.
- Use BSA as a standard protein because it has a well-defined structure and is stable in solution.
- Measure the absorbance of the samples and standard within 5 to 10 minutes after adding the Bradford reagent, as prolonged incubation can lead to a decrease in the accuracy of the assay.
- Avoid high concentrations of detergents or reducing agents in the protein sample, as these can interfere with the binding of the dye to protein and affect the accuracy of the assay.
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