Bradford Assay
Goals
- Conduct a bradford assay on the collected fraction of ADA made on 2012/11/06 and on 2012/09/26. The bradford assay will allow us to calculate the concentration of ADA present in our collected fraction samples.
- Prepare Au/HRP solutions
Procedure
- A stock solution of 1mg/mL BSA was prepared (actual concentration = 1.02mg/mL). 10.2mg BSA added to 10mL of water in a volumetric flask.
- 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.
- Note that : 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.
- After the calibration curve was made for the standards, the concentration of the ADA collected on the two days were analyzed. We ran both fracs that possessed the majority of the sample from 2012/09/26 and only frac number 2 from 2012/11/06
- In a cuvet, 7.5μL of autclaved water, 7.5 μL of a fraction and 715μL 1X bradford Reagent were added. In total 3 cuvet samples were run through the UV-Vis.
- Because the spectra for frac #8 and Frac New had an absorbance of over 1, those cuvet samples were diluted in half using the same procedure as for the standard solutions in order to get an acceptable (below 1) absorbance)
- For all information regarding the making and analyzing of the Au/HRP solutions , please see Dhea's Notebook
DATA
- Linear regression was used to determine the equation of the line
Concentrations, absorbances, for the collected fracs of ADA
| Sample Name
|
Volume of ADA[mL]
|
Absorbance
|
[ADA] [mg/mL]
|
Mass of ADA[mg]
|
Mols of ADA [mol]
|
Molar absorptivity [mL/mg*cm-1]
|
molarity[M]
|
uM
|
| Frac 2 |
0.015 |
0.93 |
0.017747193 |
0.025644694 |
6.30091E-10 |
52.40265306 |
4.20061E-05 |
42.0060507
|
| Frac 8 |
0.0075 |
0.757 |
0.011481347 |
0.016590547 |
4.0763E-10 |
65.93302839 |
5.43507E-05 |
54.35068601
|
| Frac new |
0.0075 |
0.819 |
0.013726911 |
0.019835386 |
4.87356E-10 |
59.66382586 |
6.49808E-05 |
64.98078863
|
- Frac 2 represents the flasks 2-4 collection done in September. Frac 8 represents collection of flask 1, and Frac New represents the ADA just made yesterday.
|
Project name
