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

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.
  • No alterations were made to the starter culture media protocol or the expression culture media protocol.
  • The media were then autoclaved according to the procedure. Please refer to Dhea Patel's entry for modifications to the autoclaving procedure.
  • Please refer to Keyun Wang's entry for actual masses and volumes used to make the media.

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:

• 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.