Bradford Assay Lab Report
Protein Concentration using BCA and Bradford Assays
Introduction
There are several ways to determine protein concentration for a sample such as The
Lowry Method, Bradford assay, and Bicinchoninic Acid (BCA) just to name a few. Determining the protein concentration in your sample is an important step in any laboratory workflow that involves protein extraction and/or analysis (1). Based on the assay being used, different chemicals can alter the accuracy for the results. For example, in BCA, the reducing reagents makes it reactive. Cu2+ is reduced to Cu1+ by protein in alkaline solution and the purple color change is the result of two molecules of BCA chelating to cuprous ion (2). This makes the reducing agents create a cuprous ion to chelate with the BCA, increase the absorbance readings, and skew the accuracy of the protein concentration. BCA does not react to contaminants such as detergents (2). In the Bradford assay in the readings are usually caused by elevated levels of detergent (3). When Coomassie Brilliant Blue G-250 binds to proteins, it causes a shift in the absorption maximum of the dye from 465nm (red) to 595nm (blue) in acidic solutions (2). Things that cause a change in the solution from acidic too basic will skew …show more content…
The concentrations were done by using y = mx+b. The equation used for BCA was y = .0011x +.2019. The value y represents the absorbance reading for the unknown value. When plugged into the equation x was solved for the concentration of unknown samples of BSA. The concentration for unknowns 1,2, and 3 using the BCA method were 524, 2332, 32 µg/mL. In graph 4 the standards were recorded using the Bradford method and produced a linear line and out of the line produced, a linear regression equation of y = .0004x +.5947 was produced and it was used to calculate values of unknown 1 and 2, which were 428 µg/mL, 266
Introduction
There are several ways to determine protein concentration for a sample such as The
Lowry Method, Bradford assay, and Bicinchoninic Acid (BCA) just to name a few. Determining the protein concentration in your sample is an important step in any laboratory workflow that involves protein extraction and/or analysis (1). Based on the assay being used, different chemicals can alter the accuracy for the results. For example, in BCA, the reducing reagents makes it reactive. Cu2+ is reduced to Cu1+ by protein in alkaline solution and the purple color change is the result of two molecules of BCA chelating to cuprous ion (2). This makes the reducing agents create a cuprous ion to chelate with the BCA, increase the absorbance readings, and skew the accuracy of the protein concentration. BCA does not react to contaminants such as detergents (2). In the Bradford assay in the readings are usually caused by elevated levels of detergent (3). When Coomassie Brilliant Blue G-250 binds to proteins, it causes a shift in the absorption maximum of the dye from 465nm (red) to 595nm (blue) in acidic solutions (2). Things that cause a change in the solution from acidic too basic will skew …show more content…
The concentrations were done by using y = mx+b. The equation used for BCA was y = .0011x +.2019. The value y represents the absorbance reading for the unknown value. When plugged into the equation x was solved for the concentration of unknown samples of BSA. The concentration for unknowns 1,2, and 3 using the BCA method were 524, 2332, 32 µg/mL. In graph 4 the standards were recorded using the Bradford method and produced a linear line and out of the line produced, a linear regression equation of y = .0004x +.5947 was produced and it was used to calculate values of unknown 1 and 2, which were 428 µg/mL, 266