Objective
- Prepare reagents for horseradish peroxidase (HRP) assay
- Complete HRP assay with and without gold nanoparticles to see if they interact with HRP's active site
Procedure
- HRP procedure1 was followed. Time was plotted against absorbance on the UV/Vis spectroscoper.
- 1mg horseradish peroxidase was dissolved in 1mL distilled water.
- A .2M buffer of sodium phosphate was made at a pH of 7.
- .0017M hydrogen peroxide solution was made by adding 1mL 30% hydrogen peroxide to 100mL distilled water. 1mL of hydrogen peroxide solution was diluted to 50mL with 49mL of sodium phosphate buffer for 34μM solution.
- 698μM iodophenol solution was made in 1mL DMSO, since it's not soluble in water. 2.5mM 4-aminoantipyrine (AAP) solution was prepared with the sodium phosphate buffer.
- From the stocks, solution was prepared with 18mM iodophenol, 7.285μM AAP, 1.7mM H2O2 and 115nM HRP.
- Such a solution was analyzed for the 1st, 3rd and 4th runs.
- For the 2nd run, HRP was increased to 2.3μM while AAP was kept at 7.285μM
- HRP was increased to 50μM and AAP to 156.25 μM for the final trial.
Data
Discussion
- The HRP assays were not completed today, but will be 10/3, with the reagents prepared today
References
1. Horseradish Peroxidase Assay. Gold Biotechnology File:3760-Protocol HRP AbsorbanceAssay.pdf
|