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

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  • Remake 2.5 mM luminol stock solution and 2.3 μL HRP solution to continue HRP chemiluminescence assay.
  • Make starter culture media and 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.

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.

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