UV Vis

UV-Visible Spectoscopy

UV-Vis is used to measure absorption spectroscopy in the ultraviolet-visible spectral region. We are using the UV-Vis to measure the activity of peroxidases, starting with HRP to develop a standard protocol. Eventually the UV-Vis will be used to measure the activity of the isolated ovoperoxidase from sea urchins. We used the kinetics mode of the UV-Vis to gather our data.

Steps to Run a Peroxidase through the UV-Vis

1) Turn on UV-Vis

2) Open the UV-Vis software on the computer monitor

3) Turn on Kinetics Mode

a) Set wavelength to the desired absorption (ours was 436 nm for Guaiacol)

b) Set a cycle time - the lower the cycle time the more data collected (we used 0.5 seconds)

c) Set a run time - HRP does not show significant data past a minute (so we used 60 seconds)

4) Select time-based measurements and create a name for each set of data before running the sample

5) Run a blank with DI water before running a sample

6) Fill a cuvette with the assay and place the cuvette in the UV-Vis

7)Add desired amount of peroxidase to the assay and mix with a plastic transfer pipette

8)Select the sample button on the monitor