Bradford Assay
The Bradford assay quantifies total amount or concentration of protein in a given sample. The assay utilizes Coomassie brilliant blue G-250 dye (CBBG) which binds proteins, specifically at tryptophan, tyrosine, histidine, phenylalanine, and with strongest affinity (8x more) to arginine residues. Free CBBG appears red in the cationic form and has a maximum absorbance of 470nm. Once bound to amino acids, CBBG pka shifts and exhibits a maximum abrobance of 595nm to appear blue.
The assay is monitored at 595nm after CBBG is added to bovine serum albumin (BSA) standards of known protein concentration. Absorbances of the BSA standards (dependent variable) are recorded then plotted vs. known protein concentration (independent variable). The calibration curve is corrected for the blank standard (no BSA). Increased protein concentration results in increased CBBG-protein complexes, resulting in increased absorbance at 595nm.
A line of best fit is plotted to the curve, and the equation of this line is used to determine protein concentration or mass of the experimental samples, which have been prepared with CBBG in the same manner as the BSA standards, and have had absorbance measured. The absorbance is input into y (absorbance) to solve for x (protein concentration or mass). Dilutions of BSA standards or protein sample may be necessary to ensure the protein concentration of the experimental samples fall within the range of the assay, generally 0-1mg/ml. If sample is diluted, multiply final concentration by dilution factor.
The Bradford assay quantifies total amount or concentration of protein in a given sample. The assay utilizes Coomassie brilliant blue G-250 dye (CBBG) which binds proteins, specifically at tryptophan, tyrosine, histidine, phenylalanine, and with strongest affinity (8x more) to arginine residues. Free CBBG appears red in the cationic form and has a maximum absorbance of 470nm. Once bound to amino acids, CBBG pka shifts and exhibits a maximum abrobance of 595nm to appear blue.
The assay is monitored at 595nm after CBBG is added to bovine serum albumin (BSA) standards of known protein concentration. Absorbances of the BSA standards (dependent variable) are recorded then plotted vs. known protein concentration (independent variable). The calibration curve is corrected for the blank standard (no BSA). Increased protein concentration results in increased CBBG-protein complexes, resulting in increased absorbance at 595nm.
A line of best fit is plotted to the curve, and the equation of this line is used to determine protein concentration or mass of the experimental samples, which have been prepared with CBBG in the same manner as the BSA standards, and have had absorbance measured. The absorbance is input into y (absorbance) to solve for x (protein concentration or mass). Dilutions of BSA standards or protein sample may be necessary to ensure the protein concentration of the experimental samples fall within the range of the assay, generally 0-1mg/ml. If sample is diluted, multiply final concentration by dilution factor.