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

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==Entry title==
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==Bradford Assay==
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* Insert content here...
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==Goals==
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* 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]].
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==Procedure==
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* 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.
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*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.
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Volume 1X Bradford Reagent Added [mL]'''
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| align="center" style="background:#f0f0f0;"|'''Volume BSA Stock Added [mL]'''
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| align="center" style="background:#f0f0f0;"|'''Volume Water Added [mL]'''
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| align="center" style="background:#f0f0f0;"|'''Conc. BSA [mg/mL]'''
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|-
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| 1.4||0||0.03||0
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|-
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| 1.4||0.005||0.025||0.003566434
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|-
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| 1.4||0.01||0.02||0.007132867
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|-
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| 1.4||0.015||0.015||0.010699301
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|-
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| 1.4||0.02||0.01||0.014265734
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|-
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| 1.4||0.025||0.005||0.017832168
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|-
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| 1.4||0.03||0||0.021398601
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|}
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<br>
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* The absorbance values at 595 nm of each solution were graphed versus BSA concentration in mg/mL to produce a calibration curve.
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* Using linear regression, the equation of the line was obtained.
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[[Image:Bradford_assay.png]]
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<br>
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* 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.
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* 7.5 μL of each fraction was added to 7.5 μL of autoclaved water and 715 μL of 1X Bradford reagent.
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** 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.
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Sample Name'''
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| align="center" style="background:#f0f0f0;"|'''Volume of ADA [mL]'''
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| align="center" style="background:#f0f0f0;"|'''Absorbance []'''
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| align="center" style="background:#f0f0f0;"|'''Conc. ADA [mg/mL]'''
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| align="center" style="background:#f0f0f0;"|'''Mass of ADA [mg]'''
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| align="center" style="background:#f0f0f0;"|'''Moles of ADA [mol]'''
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| align="center" style="background:#f0f0f0;"|'''Molar Absorptivity of ADA [mL/mg cm]'''
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| align="center" style="background:#f0f0f0;"|'''Molarity [M]'''
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* 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.
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==DATA==
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Revision as of 17:39, 11 November 2012

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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]
1.400.030
1.40.0050.0250.003566434
1.40.010.020.007132867
1.40.0150.0150.010699301
1.40.020.010.014265734
1.40.0250.0050.017832168
1.40.0300.021398601


  • 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

  • Fractions of ADA, made on 2012/10/03 and 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.
Sample Name Volume of ADA [mL] Absorbance [] Conc. ADA [mg/mL] Mass of ADA [mg] Moles of ADA [mol] Molar Absorptivity of ADA [mL/mg cm] 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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