Bradford Assay
Goals
Procedure
- 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]
|
| 1.4 |
0 |
0.03 |
0
|
| 1.4 |
0.005 |
0.025 |
0.003566434
|
| 1.4 |
0.01 |
0.02 |
0.007132867
|
| 1.4 |
0.015 |
0.015 |
0.010699301
|
| 1.4 |
0.02 |
0.01 |
0.014265734
|
| 1.4 |
0.025 |
0.005 |
0.017832168
|
| 1.4 |
0.03 |
0 |
0.021398601
|
- 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.
| Sample Name
|
Volume of ADA [mL]
|
Absorbance []
|
Conc. ADA [mg/mL]
|
Mass of ADA [mg]
|
Moles of ADA [mol]
|
Molar Absorptivity of ADA [mL/mg cm]
|
Molarity [M]
- 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.
DATA
|
|
Project name
