User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/07

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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] 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.

DATA

Bradford assay.png

  • Linear regression was used to determine the equation of the line


  • 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