- A stock solution of 1mg/mL BSA was prepared by adding 10mg of BSA into a 10mL volumetric flask and then filling it with autoclaved water. The actual concentration of the BSA stock was 1.02mg/mL as the actual weighed out BSA added was 10.2mg BSA.
- In order to determine the concentration of the ADA collected in the fracs, standard solutions had to be made and run through the UV-Vis first. Seven standard solutions were prepared with 1X Bradford Reagent, varying amounts of BSA stock, and autoclaved water. These solutions were analyzed in the UV-Vis from 500-800 nm.
|Volume 1X Bradford Reagent Added [mL]
||Volume BSA Stock Added [mL]
||Volume Water Added [mL]
||Conc. BSA [mg/mL]
- The absorbance values at 595 nm of each solution were graphed versus BSA concentration in mg/mL to produce a calibration curve.
- Using linear regression, the equation of the line was obtained.
- Fractions of ADA, made on 2012/10/03 and 2012/11/06, were obtained.
- 7.5 μL of each fraction was added to 7.5 μL of autoclaved water and 715 μL of 1X Bradford reagent.
- Fraction #2, obtained in November, was diluted by half, using the same procedure as listed above for the standard solutions, such that its absorbance was below 1.
||Volume of ADA [mL]
||Conc. ADA [mg/mL]
||Mass of ADA [mg]
||Moles of ADA [mol]
||Molar Absorptivity of ADA [mL/mg cm]
- When these samples were originally run, they produced absorbances over 1 which resulted in us needing to dilute the solution further. As a result, all solutions were diluted by a half to 715μL of original solution with an additional 715μL of water.