< User:Melissa Novy | Notebook | CHEM-571 | 2012 | 09
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- Complete horseradish peroxidase enzymatic activity assay from 2012/09/18.
- Make 2.5 mM luminol (MW 177.16) stock solution for horseradish peroxidase chemiluminescence assay.
- Using a fluorimeter, collect emission spectra at 425 nm for horseradish peroxidase chemiluminescence assay.
Luminol Stock Solution in H2O and Carbonate Buffer
- 10 mL of 2.5 mM luminol stock solution requires the following:
MW luminol: 177.16 g/mol
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
0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10-3 M in H2O
- The concentration of the luminol stock solution was varied by serial dilution as follows:
- 1.25 mM, 625 μM, 312.5 μM, and 156.25 μM.
- 10 mL solutions of each concentration were made.
- Luminol did not dissolve completely in H2O, so Na2CO3 was added in a ratio of 4 g for every 500 mL of solution to aid its dissolution.
- 0.08 g of Na2CO3 was added to the luminol solution.
- After the luminol dissolved, 0.48 g NaHCO3 was added to adjust the pH of the solution to about 7.
- 0.48 g of NaHCO3 was added to the luminol solution.
- Please refer to the entry on 2012/09/25 for HRP-luminol fluorescence spectra and data analysis.