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

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(HRP Chemiluminescence Assay)
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* It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values.  To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph).  As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.
* It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values.  To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph).  As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.
* The HRP concentration was further varied from trials on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|2012/09/25]] in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.
* The HRP concentration was further varied from trials on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|2012/09/25]] in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.
-
* ???
+
* Suggestions to produce better results are to alter the pH and to add H<sub>2</sub>O<sub>2</sub> to begin the reaction instead of HRP.
==Notes==
==Notes==

Revision as of 12:28, 18 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.
  • 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?

Binding and Elution Buffers

  • Note that these buffers will be used to purify the proteins extracted from E. coli to ultimately obtain adenosine deaminase.
  • After the buffers were made, the pH of each was adjusted to 7.5 by adding 1 N HCl dropwise and monitoring the pH with a Thermo Scientific Orion 5 Star pH meter.
  • 1 L of binding buffer was made with components of imidazole, Tris, and NaCl at the following concentrations in 1 L of water:
    • 0.03 M imidazole
    • 0.02 M Tris
    • 0.5 M NaCl
  • 500 mL of elution buffer was made with components of imidazole, Tris, and NaCl at the following concentrations in 500 mL of water:
    • 0.5 M imidazole
    • 0.02 M Tris
    • 0.5 M NaCl

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

  • Solutions made on 2012/09/25 were used to continue the HRP-luminol 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


  • Initial and final concentrations (in a total volume of ~2 mL) of all HRP-luminol reaction solutions that gave usable results:
' Initial Concentrations [mM] ' ' ' Final Concentrations [mM] ' ' '
Trial4-IodophenolLuminolH2O2HRP4-IodophenolLuminolH2O2HRP
1181.251.70.00230.0001190.0005780.0008467.63E-08
2180.6251.70.00230.0001190.0002890.0008467.63E-08
3180.6250.850.00230.0001190.0002890.0004237.63E-08
4181.250.4250.00230.0001190.0005780.0002127.63E-08
5181.251.70.00230.0001190.0005780.0008467.63E-08
6181.251.70.001150.0001190.0005780.0008463.82E-08


Image:HRP_Chemiluminescence_Assay.JPG

  • Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.
  • It was observed that the fluorescence spectra obtained from HRP-luminol assay solutions were producing negative intensity values. To determine why this was the case, a blank of carbonate buffer was also analyzed (see graph). As this blank also produced negative intensity values, it was determined that the baseline was negative and some fluorescence was actually being produced by the assay solutions.
  • The HRP concentration was further varied from trials on 2012/09/25 in an attempt to slow the reaction rate such that the fluorimeter could detect the decline in fluorescence.
  • Suggestions to produce better results are to alter the pH and to add H2O2 to begin the reaction instead of HRP.

Notes

  • The volumes of the components added to each of the reaction solutions was not varied during the experiment. Please refer to the following table for the volumes added:
Solution Volume [μL]
4-Iodophenol13.2
Luminol920
H2O2990
HRP66
  • These volumes were used to calculate the final concentrations of the solution components in the assay solutions.


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