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

Bradford Assay

Goals

• Conduct a bradford assay on the collected fraction of ADA made on 2012/11/06 and on 2012/09/26.
• Prepare Au/HRP solutions

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.

• 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 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