User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/07: Difference between revisions
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== | ==Bradford Assay== | ||
==Goals== | |||
* Conduct a bradford assay on the collected fraction of ADA made on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/06| 2012/11/06]] and on [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/26| 2012/09/26]]. | |||
==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. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Volume 1X Bradford Reagent Added [mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume BSA Stock Added [mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume Water Added [mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
<br> | |||
* The absorbance values at 595 nm of each solution were graphed versus BSA concentration in mg/mL to produce a calibration curve. | |||
* Using linear regression, the equation of the line was obtained. | |||
[[Image:Bradford_assay.png]] | |||
<br> | |||
* Fractions of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|2012/11/06]], were obtained. | |||
* 7.5 μL of each fraction was added to 7.5 μL of autoclaved water and 715 μL of 1X Bradford reagent. | |||
** Fraction #2, obtained in November, was diluted by half, using the same procedure as listed above for the standard solutions, such that its absorbance was below 1. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample Name''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume of ADA [mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Absorbance []''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. ADA [mg/mL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Mass of ADA [mg]''' | |||
| align="center" style="background:#f0f0f0;"|'''Moles of ADA [mol]''' | |||
| align="center" style="background:#f0f0f0;"|'''Molar Absorptivity of ADA [mL/mg cm]''' | |||
| align="center" style="background:#f0f0f0;"|'''Molarity [M]''' | |||
* 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== | |||
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Revision as of 14:39, 11 November 2012
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Bradford AssayGoals
Procedure
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