Answer to Question #123844 in Biochemistry for Ashu

Question

The BSA standard solution with the concentration of 0.5 mg/mL was used to produce standard curve for protein assay based on Bradford method. The test tubes contain BSA solutions was prepared: 0, 20, 40, 60, 80, 100 μL. The reading of the absorbance for each test tube after adding the Bradford reagent was 0, 0.05, 0.09, 0.14, 0.19 and 0.22. If 20 μL of protein solution with unknown concentration were taken and mixed with 80 μL water. Then, 10 μL of this mixture was reacted with Bradford reagent and the absorbance reading was 0.08. What is unknown protein solution concentration before dilution?

Answer 1

Assuming that the standard solutions of BSA were made upto 100μL, the concentrations taken from the working standard solution of 0.5 mg/mL would be 0, 100, 200, 300 , 400 & 500 μg/mL, respectively. Using an X-Y plot, with concentration on X axis & absorbance on Y axis, the standard equation would be y = 0.0004x + 0.0029, with an R squared value of 0.9961. Given that, 20 μL of the unknown sample was made upto 100μL and the absorbance recorded for 10μL was 0.08. Substituting the given absorbance in the standard equation and solving for x; we will obtain 192.75 μg/mL as the concentration after dilution. Multiplying by the dilution factor of 5 (20μL diluted to 100μL), the unknown concentration before dilution will be 192.75*5=963.75μg/mL (assuming the volume taken for reading the absorbance of known concentration as 10μL).