User:Melissa Novy/Notebook/CHEM-571/2012/09/26

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(HRP Chemiluminescence Assay)
(Objectives)
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** Make binding buffer and elution buffer.
** Make binding buffer and elution buffer.
** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26|Dhea Patel's entry]] for the protocols and calculations of the above solutions.
** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26|Dhea Patel's entry]] for the protocols and calculations of the above solutions.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need ot include the specifics of the bidnign nad elution bufers, pH final volume and concnetratiosn of each component.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:09, 7 October 2012 (EDT)''':you need to include the specifics of the binding and elution buffers, pH final volume and concentration of each component.
* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
* Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
* Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:14, 7 October 2012 (EDT)''':didn't you also need ot do something with the cells for making the ADA protein?
==Spectroscopy Data of Tris Buffers==
==Spectroscopy Data of Tris Buffers==

Revision as of 18:14, 7 October 2012

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Objectives

  • Prepare cells for protein expression.
    • Make binding buffer and elution buffer.
    • Please refer to Dhea Patel's entry for the protocols and calculations of the above solutions.
  • Abigail E. Miller 18:09, 7 October 2012 (EDT):you need to include the specifics of the binding and elution buffers, pH final volume and concentration of each component.
  • Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
  • Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.
  • Abigail E. Miller 18:14, 7 October 2012 (EDT):didn't you also need ot do something with the cells for making the ADA protein?

Spectroscopy Data of Tris Buffers

Image:Tris_Buffer_Absorbance.JPG

  • Graph of absorbance versus wavelength of Tris buffer in H2O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
  • Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.


  • Abigail E. Miller 18:11, 7 October 2012 (EDT):it is not at all clear that this is tris buffer added to AuNPs. and there is no link to where the AuNPs came from or when these solutions were mixed. or is that not what this is about?

HRP Chemiluminescence Assay

  • Concentrations of solutions tested:
Trial Phenol Luminol H2O2 HRP
118 mM1.25 mM1.7 mM0.23 µM
218 mM0.625 mM1.7 mM0.23 µM
318 mM1.25 mM1.7 mM9.2 µM
418 mM1.25 mM1.7 mM4.6 µM


Image:HRP_Chemiluminescence_Assay.JPG

  • Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.
  • Refer to the report for analysis of HRP assays with AAP and luminol.
  • Abigail E. Miller 18:12, 7 October 2012 (EDT):again, included all information in your notebook. everything in the report should be pulled form your notebook. also , where di the stock solutions come from?


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