Abbreviations:

• mcg = micrograms
• mcL = microliters
• BSA = bovine serum albumin
• O.D. = optical density
• dI = deionized

Background

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).

Procedure

• 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, mcL	Vol. Water, mcL	Vol. Bradford Reagent, mcL
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