User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/19

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#To determine enzymatic activity of horseradish peroxidase via the usage of fluorometer under 510nm.
#To determine enzymatic activity of horseradish peroxidase via the usage of fluorometer under 510nm.
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==Description==
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*The stock solutions of HRP, H<sub>2</sub>O<sub>2</sub>, and phenol that were made [http://openwetware.org/wiki/User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/09/18 yesterday] were mixed and run under the fluorometer at 510nm.
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*10mL of 2.5mM Luminol stock was prepared using the following calculations:
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**'''MW luminol''': 177.16 g/mol
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**'''Mass luminol''':
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        10 mL × (10<sup>-3</sup> L/mL) × (2.5 × 10<sup>-3</sup> M) × (10<sup>-3</sup> mol/mmol) × 177.16 g/mol = 0.00443 g luminol
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**'''Actual mass luminol''': 0.0044 g
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**'''Actual concentration''':
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        0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10<sup>-3</sup> M in H<sub>2</sub>O
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***Because the luminol did not completely dissolve in H<sub>2</sub>O, 0.08g Na<sub>2</sub>CO<sub>3</sub> was added to aid in its dissolution.
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****Ratio: 4g sodium carbonate/500mL solution
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***After the luminol dissolved with the sodium carbonate, 0.48g Na<sub>2</sub>HCO<sub>3</sub> was added to the solution to neutralize the pH of the solution to about pH 7.
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*The first sample was mixed with 4-Iodophenol, H<sub>2</sub>O<sub>2</sub>, luminol, and HRP. Please refer to [User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/09/19 Kay's entry] for details in initial concentration, final concentration, and volumes added.
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Revision as of 13:22, 25 September 2012

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Objective

  1. To determine enzymatic activity of horseradish peroxidase via the usage of fluorometer under 510nm.

Description

  • The stock solutions of HRP, H2O2, and phenol that were made yesterday were mixed and run under the fluorometer at 510nm.
  • 10mL of 2.5mM Luminol stock was prepared using the following calculations:
    • MW luminol: 177.16 g/mol
    • Mass luminol: 
        10 mL × (10-3 L/mL) × (2.5 × 10-3 M) × (10-3 mol/mmol) × 177.16 g/mol = 0.00443 g luminol
    • Actual mass luminol: 0.0044 g
    • Actual concentration: 
        0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10-3 M in H2O
      • Because the luminol did not completely dissolve in H2O, 0.08g Na2CO3 was added to aid in its dissolution.
        • Ratio: 4g sodium carbonate/500mL solution
      • After the luminol dissolved with the sodium carbonate, 0.48g Na2HCO3 was added to the solution to neutralize the pH of the solution to about pH 7.
  • The first sample was mixed with 4-Iodophenol, H2O2, luminol, and HRP. Please refer to [User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/09/19 Kay's entry] for details in initial concentration, final concentration, and volumes added.


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