Bradford Protein Concentration Assay
- mcg = micrograms
- mcL = microliters
- BSA = bovine serum albumin
- O.D. = optical density
- dI = deionized
The Bradford protein assay (1) is one of several simple methods commonly used to determine the total protein concentration of a sample. The method is based on the proportional binding of the dye Coomassie to proteins. Within the linear range of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker. Coomassie absorbs at 595 nm. The protein concentration of a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in this assay. Although different protein standards can be used, we have chosen the most widely used protein as our standard - Bovine Serum Albumin (BSA).
- Prepare a 4-fold dilution of a 2 mg/mL BSA sample by adding 50 mcL of 2 mg/mL BSA to 150 mcL of dI water to make 200 mcL of 0.5 mg/mL BSA.
- Generate test samples for the reference cell, blank, BSA standards and the protein sample to be tested according to Table 1 in disposable cuvettes.
- Note that the "reference cell" and "blank" are identical. A reference cell test sample is only required when using a double-beam UV-visible spectrophotometer for absorbance measurements.
- Note that a dilution of the protein sample may be required for the resulting absorbance to fall within the linear range of the assay.
- Allow each sample to incubate at room temperature for 10-30 minutes. (Record the actual incubation time in your notebook.)
- Measure the absorbance of each sample at 595 nm using a UV-visible spectrophotometer. Be sure to allow the instrument to warm up for at least 15 minutes prior to use.
- Plot the absorbance of each BSA standard as a function of its theoretical concentration. The plot should be linear.
- Determine the best fit of the data to a straight line in the form of the equation "y = mx + b" where y = absorbance at 595 nm and x = protein concentration.
- Use this equation to calculate the concentration of the protein sample based on the measured absorbance. Note: If the absorbance of the test sample is outside of the absorbance range for the standards, then the assay must be repeated with a more appropriate dilution, if any. The linear range for the assay (and for most spectrophotometers is 0.2 - 0.8 O.D. units.
Table 1. Preparation of test samples for the Bradford protein assay.
| Test Sample || Sample Volume, |
| Vol. Water, |
| Vol. Bradford Reagent, |
| Reference Cell || 0 || 800 || 200 |
| Blank || 0 || 800 || 200 |
| BSA Standard - 5 mcg/mL || 10 || 790 || 200 |
| BSA Standard - 10 mcg/mL || 20 || 780 || 200 |
| BSA Standard - 15 mcg/mL || 30 || 770 || 200 |
| BSA Standard - 20 mcg/mL || 40 || 760 || 200 |
| BSA Standard - 25 mcg/mL || 50 || 750 || 200 |
| Protein Sample || 50 || 750 || 200 |