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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]
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Volume BSA Stock Added [mL]
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Volume Water Added [mL]
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Conc. BSA [mg/mL]
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| 1.4 |
0 |
0.03 |
0
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| 1.4 |
0.005 |
0.025 |
0.003566434
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| 1.4 |
0.01 |
0.02 |
0.007132867
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| 1.4 |
0.015 |
0.015 |
0.010699301
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| 1.4 |
0.02 |
0.01 |
0.014265734
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| 1.4 |
0.025 |
0.005 |
0.017832168
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| 1.4 |
0.03 |
0 |
0.021398601
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- 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
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Volume of ADA [mL]
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Absorbance []
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Conc. ADA [mg/mL]
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Mass of ADA [mg]
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Moles of ADA [mol]
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Molar Absorptivity of ADA [mL/mg cm]
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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
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