User:Michael F. Nagle/Notebook/Chem 571/2012/11/07

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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) 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
20 10 0.15 2.26E-06
25 5 0.24 3.61E-06
30 0 1 1.51E-05


  • 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

Bradforassay.png

Fraction # Absorbance [ADA] [mg/mL] molar absorptivity [mL/mg*cm-1] Molarity (µM)
2 0.93 0.0177 52.4 42.0
8 0.757 0.0115 65.9 54.4
9 0.819 0.0137 59.7 65.0


Frac 2 was found to be 42µM, while 8 was 54.4µM and 9 was 65µM.