User:Melissa Novy/Notebook/CHEM-571/2012/09/25

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(Horseradish Peroxidase Chemiluminescence Assay)
Current revision (11:18, 9 October 2012) (view source)
(Horseradish Peroxidase Chemiluminescence Assay)
 
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==Objectives==
==Objectives==
* Remake 2.5 mM luminol stock solution and 2.3 μL HRP solution to continue HRP chemiluminescence assay.
* Remake 2.5 mM luminol stock solution and 2.3 μL HRP solution to continue HRP chemiluminescence assay.
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* Make starter culture media and expression culture media to use with <i>E. coli</i> cells expressing adenosine deaminase enzyme.
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* Make four sets of 1 L each of starter culture media and four sets of 1 L each of expression culture media to use with <i>E. coli</i> cells expressing adenosine deaminase enzyme.
** No alterations were made to the [[AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media|starter culture media protocol]] or the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media|expression culture media protocol]].
** No alterations were made to the [[AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media|starter culture media protocol]] or the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media|expression culture media protocol]].
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** The media were then autoclaved according to the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave|procedure]].
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** The media were then autoclaved according to the [[AU_Biomaterials_Design_Lab:Protocols/Autoclave|procedure]].  Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/25|Dhea Patel's entry]] for modifications to the autoclaving procedure.
** Please refer to [[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/09/25|Keyun Wang's entry]] for actual masses and volumes used to make the media.
** Please refer to [[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/09/25|Keyun Wang's entry]] for actual masses and volumes used to make the media.
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==Horseradish Peroxidase Stock Solution in H<sub>2</sub>O==
==Horseradish Peroxidase Stock Solution in H<sub>2</sub>O==
* 1 mL of 23 μM HRP solution was made by adding 0.0010 g of HRP to 1 mL of H<sub>2</sub>O.
* 1 mL of 23 μM HRP solution was made by adding 0.0010 g of HRP to 1 mL of H<sub>2</sub>O.
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* The solution was diluted to concentrations of 2.3 μM, 1.15 μM, 0.23 μM, and 23 nM by serial dilution.
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* The solution was diluted to concentrations of 2.3 μM, 1.15 μM, 0.23 μM, and 23 nM by serial dilution.  For each solution, 1 mL of HRP solution was added to 9 mL of H<sub>2</sub>O.
==Horseradish Peroxidase Chemiluminescence Assay==
==Horseradish Peroxidase Chemiluminescence Assay==
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[[Image:HRP_Assay_2012-09-25.png]]
[[Image:HRP_Assay_2012-09-25.png]]
* Graph of intensity versus time of <i>p</i>-iodophenol-enhanced solutions of luminol, H<sub>2</sub>O<sub>2</sub>, and horseradish peroxidase.
* Graph of intensity versus time of <i>p</i>-iodophenol-enhanced solutions of luminol, H<sub>2</sub>O<sub>2</sub>, and horseradish peroxidase.
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* Please refer to the above table for initial concentrations of the components.  Final concentrations will be calculated in the future.
+
* Please refer to the above table for initial concentrations of the components.  Final concentrations of the components in the ~2 mL final volume of the reaction solution will be calculated in the future.
* Based on the shape of the curves, it appears that the reaction is proceeding too quickly upon the addition of HRP, such that the slope is too steep.  It is recommended that the concentration of HRP be decreased, while the concentration of luminol to H<sub>2</sub>O<sub>2</sub> optimized.
* Based on the shape of the curves, it appears that the reaction is proceeding too quickly upon the addition of HRP, such that the slope is too steep.  It is recommended that the concentration of HRP be decreased, while the concentration of luminol to H<sub>2</sub>O<sub>2</sub> optimized.

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Objectives

  • Remake 2.5 mM luminol stock solution and 2.3 μL HRP solution to continue HRP chemiluminescence assay.
  • Make four sets of 1 L each of starter culture media and four sets of 1 L each of expression culture media to use with E. coli cells expressing adenosine deaminase enzyme.

Luminol Stock Solution in H2O and Carbonate Buffer

  • Please refer to entry on 2012/09/19 for luminol stock solution protocol and calculations.
    • Actual mass luminol: 0.00443 g
    • Actual concentration luminol: 2.5 mM
    • Actual mass Na2CO3: 0.08 g
    • Actual mass NaHCO3: 0.48 g

Horseradish Peroxidase Stock Solution in H2O

  • 1 mL of 23 μM HRP solution was made by adding 0.0010 g of HRP to 1 mL of H2O.
  • The solution was diluted to concentrations of 2.3 μM, 1.15 μM, 0.23 μM, and 23 nM by serial dilution. For each solution, 1 mL of HRP solution was added to 9 mL of H2O.

Horseradish Peroxidase Chemiluminescence Assay

  • Note that the HRP solution was further diluted (see above) in an attempt to slow the reaction rate such that the reaction progress could be observed via fluorescence.
  • Other studies on HRP chemiluminescence assays indicated that the optimal mole ratio of H2O2 to luminol is about 2.2.
  • The intensity of the chemiluminescence of the solutions was recorded over a period of 300 s. The excitation wavelength was 350 nm and the emission wavelength was 430 nm. The excitation slit width was 15 nm and the emission slit width was 20 nm.
    • The baseline for the solutions was established by measuring the intensity of a solution of sodium carbonate buffer in water.


  • The initial concentrations of the components of the chemiluminescence assay solutions are as follows:
Trial Phenol Luminol H2O2 HRP
118 mM1.25 mM425 µM2.3 µM
218 mM1.25 mM1.7 mM2.3 µM
318 mM1.25 mM1.7 mM1.15 µM


Image:HRP_Assay_2012-09-25.png

  • Graph of intensity versus time of p-iodophenol-enhanced solutions of luminol, H2O2, and horseradish peroxidase.
  • Please refer to the above table for initial concentrations of the components. Final concentrations of the components in the ~2 mL final volume of the reaction solution will be calculated in the future.
  • Based on the shape of the curves, it appears that the reaction is proceeding too quickly upon the addition of HRP, such that the slope is too steep. It is recommended that the concentration of HRP be decreased, while the concentration of luminol to H2O2 optimized.



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