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Objective
- Determine concentration of ADA prepared Oct. 23 via Bradford Assay
Bradford Assay
- Solutions were prepared with 715μL 1x Bio-Rad Bradford reagant and the following concentrations of BSA:
| volume BSA (uL)
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volume H2O (uL
|
[BSA] (mg/mL)
|
[BSA] (M)
|
| 0 |
30 |
0 |
0.00E+00
|
| 5 |
25 |
0.24 |
3.61E-06
|
| 10 |
20 |
0.15 |
2.26E-06
|
| 15 |
15 |
0.13 |
2.01E-06
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| 20 |
10 |
0.15 |
2.26E-06
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| 25 |
5 |
0.24 |
3.61E-06
|
| 30 |
0 |
1 |
1.51E-05
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- A UV/Vis calibration curve was obtained by analyzing the samples and plotting BSA concentration against absorbance at 595nm.
- The ADA purified yesterday, labelled Frac 9, was analyzed along with Fracs 2 and 8 purified 9/26 via UV/Vis and it's peak was compared to the linear regression of BSA's in order to determine concentration.
Data
| Fraction #
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Absorbance
|
[ADA] [mg/mL]
|
molar absorptivity [mL/mg*cm-1]
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Molarity (µM)
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| 2 |
0.93 |
0.0177 |
52.4 |
42.0
|
| 8 |
0.757 |
0.0115 |
65.9 |
54.4
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| 9 |
0.819 |
0.0137 |
59.7 |
65.0
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Frac 2 was found to be 42µM, while 8 was 54.4µM and 9 was 65µM.
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