Bradford Protein Concentration Assay

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Bradford Protein Concentration Assay version 01/07/2001 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 处理 SSI 文件时出错