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

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(Objectives)
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* 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.
 +
 +
==Cell Protein Expression==
 +
* Protocol
 +
*# The four inoculated starter cultures, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|2012/09/25]] were centrifuged in a Sorvali RC6+ centrifuge for 15 min at 4°C and at 4500 rpm.
 +
*# The supernatant was discarded and the four resultant <i>E. coli</i> cell pellets were resuspended in 4 mL each of luria broth from the expression cultures, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|2012/09/25]].
 +
*# The resuspended solutions were added to the expression cultures and the flasks were incubated and shaken at 37°C and 160 rpm.
 +
*# 1 mL of 0.4 M IPTG was added to each of the flasks.  Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/26|Dhea Patel's entry]] for how the IPTG solution was made.
 +
*# Incubation and shaking of the flasks was resumed for 3 hours.
 +
*# The solutions were centrifuged at 4500 rpm and at 4°C for a period of 15 min to pellet the cells.
 +
*# The supernatant was discarded and the cell pellets were resuspended in 30 mL of binding buffer, made today, to give a total of four suspended cell solutions.
 +
*# The solutions were stored at -80°C.
 +
 +
==Binding and Elution Buffers==
 +
* Note that these buffers will be used to purify the proteins extracted from <i>E. coli</i> 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==
==Spectroscopy Data of Tris Buffers==
[[Image:Tris_Buffer_Absorbance.JPG]]
[[Image:Tris_Buffer_Absorbance.JPG]]
* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
* Graph of absorbance versus wavelength of Tris buffer in H<sub>2</sub>O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
 +
** The Tris buffers were made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/04|2012/09/04]].
* 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.
* 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.
 +
 +
==HRP Chemiluminescence Assay==
 +
* Solutions made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/25|2012/09/25]] were used to continue the HRP-luminol assay.
 +
* Concentrations of solutions tested:
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Trial'''
 +
| align="center" style="background:#f0f0f0;"|'''Phenol'''
 +
| align="center" style="background:#f0f0f0;"|'''Luminol'''
 +
| align="center" style="background:#f0f0f0;"|'''H2O2'''
 +
| align="center" style="background:#f0f0f0;"|'''HRP'''
 +
|-
 +
| 1||18 mM||1.25 mM||1.7 mM||0.23 µM
 +
|-
 +
| 2||18 mM||0.625 mM||1.7 mM||0.23 µM
 +
|-
 +
| 3||18 mM||1.25 mM||1.7 mM||9.2 µM
 +
|-
 +
| 4||18 mM||1.25 mM||1.7 mM||4.6 µM
 +
|}
 +
<br>
 +
* Initial and final concentrations (in a total volume of ~2 mL) of all HRP-luminol reaction solutions that gave usable results:
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|'''Initial Concentrations [mM]'''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|'''Final Concentrations [mM]'''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
|-
 +
| Trial||4-Iodophenol||Luminol||H<sub>2</sub>O<sub>2</sub>||HRP||4-Iodophenol||Luminol||H<sub>2</sub>O<sub>2</sub>||HRP
 +
|-
 +
| 1||18||1.25||1.7||0.0023||0.000119||0.000578||0.000846||7.63E-08
 +
|-
 +
| 2||18||0.625||1.7||0.0023||0.000119||0.000289||0.000846||7.63E-08
 +
|-
 +
| 3||18||0.625||0.85||0.0023||0.000119||0.000289||0.000423||7.63E-08
 +
|-
 +
| 4||18||1.25||0.425||0.0023||0.000119||0.000578||0.000212||7.63E-08
 +
|-
 +
| 5||18||1.25||1.7||0.0023||0.000119||0.000578||0.000846||7.63E-08
 +
|-
 +
| 6||18||1.25||1.7||0.00115||0.000119||0.000578||0.000846||3.82E-08
 +
|}
 +
<br>
 +
[[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 [[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==
 +
* 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:
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Solution'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume [μL]'''
 +
|-
 +
| 4-Iodophenol||13.2
 +
|-
 +
| Luminol||920
 +
|-
 +
| H<sub>2</sub>O<sub>2</sub>||990
 +
|-
 +
| HRP||66
 +
|}
 +
* These volumes were used to calculate the final concentrations of the solution components in the assay solutions.
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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.

Cell Protein Expression

  • Protocol
    1. The four inoculated starter cultures, made on 2012/09/25 were centrifuged in a Sorvali RC6+ centrifuge for 15 min at 4°C and at 4500 rpm.
    2. The supernatant was discarded and the four resultant E. coli cell pellets were resuspended in 4 mL each of luria broth from the expression cultures, made on 2012/09/25.
    3. The resuspended solutions were added to the expression cultures and the flasks were incubated and shaken at 37°C and 160 rpm.
    4. 1 mL of 0.4 M IPTG was added to each of the flasks. Please refer to Dhea Patel's entry for how the IPTG solution was made.
    5. Incubation and shaking of the flasks was resumed for 3 hours.
    6. The solutions were centrifuged at 4500 rpm and at 4°C for a period of 15 min to pellet the cells.
    7. The supernatant was discarded and the cell pellets were resuspended in 30 mL of binding buffer, made today, to give a total of four suspended cell solutions.
    8. The solutions were stored at -80°C.

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.

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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