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

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(Procedure)
Current revision (14:51, 24 November 2012) (view source)
(DATA)
 
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==Goals==
==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]].  
+
* 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]]. The bradford assay will allow us to calculate the concentration of ADA present in our collected fraction samples.
 +
*Prepare Au/HRP solutions
==Procedure==
==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.  
+
* A stock solution of 1mg/mL BSA was prepared (actual concentration = 1.02mg/mL).  10.2mg BSA added to 10mL of water in a volumetric flask.  
*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.  
*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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* The absorbance values at 595 nm of each solution were graphed versus BSA concentration in mg/mL to produce a calibration curve.
* 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 [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/26| 2012/09/26]] and only frac number 2 from [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/06| 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 making and analyzing of the Au/HRP solutions , please see [[User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/11/07| Dhea's Notebook]]
==DATA==
==DATA==
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* Linear regression was used to determine the equation of the line
* Linear regression was used to determine the equation of the line
 +
Concentrations, absorbances, for the collected fracs of ADA
-
* 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.
+
{| {{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;"|'''[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;"|'''Mols of ADA [mol]'''
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| align="center" style="background:#f0f0f0;"|'''Molar absorptivity [mL/mg*cm-1]'''
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| align="center" style="background:#f0f0f0;"|'''molarity[M]'''
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| align="center" style="background:#f0f0f0;"|'''uM'''
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|-
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| Frac 2||0.015||0.93||0.017747193||0.025644694||6.30091E-10||52.40265306||4.20061E-05||42.0060507
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|-
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| Frac 8||0.0075||0.757||0.011481347||0.016590547||4.0763E-10||65.93302839||5.43507E-05||54.35068601
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|-
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| Frac new||0.0075||0.819||0.013726911||0.019835386||4.87356E-10||59.66382586||6.49808E-05||64.98078863
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|}
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==DATA==
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** Frac 2 represents the flasks 2-4 collection done in [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/26| September]]. Frac 8 represents collection of [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/26|flask 1]], and Frac New represents the ADA just made [[User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/06| yesterday.]]
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Bradford Assay

Goals

  • Conduct a bradford assay on the collected fraction of ADA made on 2012/11/06 and on 2012/09/26. The bradford assay will allow us to calculate the concentration of ADA present in our collected fraction samples.
  • Prepare Au/HRP solutions

Procedure

  • A stock solution of 1mg/mL BSA was prepared (actual concentration = 1.02mg/mL). 10.2mg BSA added to 10mL of water in a volumetric flask.
  • 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.
  • 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 making and analyzing of the Au/HRP solutions , please see Dhea's Notebook

DATA

Image:Bradford_assay.png

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

Concentrations, absorbances, for the collected fracs of ADA

Sample Name Volume of ADA[mL] Absorbance [ADA] [mg/mL] Mass of ADA[mg] Mols of ADA [mol] Molar absorptivity [mL/mg*cm-1] molarity[M] uM
Frac 20.0150.930.0177471930.0256446946.30091E-1052.402653064.20061E-0542.0060507
Frac 80.00750.7570.0114813470.0165905474.0763E-1065.933028395.43507E-0554.35068601
Frac new0.00750.8190.0137269110.0198353864.87356E-1059.663825866.49808E-0564.98078863
    • Frac 2 represents the flasks 2-4 collection done in September. Frac 8 represents collection of flask 1, and Frac New represents the ADA just made yesterday.


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