Difference between revisions of "Peroxidase UV Vis assay"
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| − | + | This research project involved the oxidation of guaiacol using the reaction between horseradish peroxidase and hydrogen peroxide. The purpose of this project was to develop a protocol for measuring the activity of a peroxidase using the UV-Vis. | |
| + | |||
==Standard Mixing Procedure== | ==Standard Mixing Procedure== | ||
| − | The mixture of Peroxidase UV-Vis assays requires the use of a Guaiacol stock solution and a hydrogen peroxide stock solution. The stock solutions are prepared as follows: | + | The mixture of Peroxidase UV-Vis assays requires the use of a Guaiacol stock solution and a hydrogen peroxide stock solution. |
| + | |||
| + | The stock solutions are prepared as follows: | ||
| − | Guaiacol Stock Solution | + | '''Guaiacol Stock Solution''' |
49.5 microliters of Guaiacol is added to a 25 mL pH 8.0 buffer solution, which is comprised of 25 mL of DI water and a 8.0 pH buffer tablet that has been mixed thoroughly. | 49.5 microliters of Guaiacol is added to a 25 mL pH 8.0 buffer solution, which is comprised of 25 mL of DI water and a 8.0 pH buffer tablet that has been mixed thoroughly. | ||
| − | Hydrogen Peroxide Stock Solution | + | '''Hydrogen Peroxide Stock Solution''' |
17 microliters of hydrogen peroxide is added to 1 mL of DI water. | 17 microliters of hydrogen peroxide is added to 1 mL of DI water. | ||
| − | Assay | + | '''Assay''' |
5.0 mL Guaiacol stock solution is added to a vial. | 5.0 mL Guaiacol stock solution is added to a vial. | ||
| Line 19: | Line 22: | ||
The desired amount of peroxidase is then added to the assay before the UV-Vis is ran. | The desired amount of peroxidase is then added to the assay before the UV-Vis is ran. | ||
| − | == | + | ==References== |
| + | [[Media:J._Biol._Chem.-1984-Deits-13525-33.pdf|Purification and Assay publication]] | ||
| + | |||
| + | ==Progress as of December 2018== | ||
| + | |||
| + | The main focus of the first semester of this research project has been to develop an efficient lab protocol for measuring the activity of a peroxidase. Since we will eventually be working with sea urchin peroxidase, background research was done on how to properly care for sea urchins as well as how to make UV-Vis assays for sea urchin peroxidase. With this information, we developed a standard mixing protocol for the UV-Vis assay using HRP. Several weeks have been spent modifying this procedure to produce more consistent results. Brad has taught me how to use the kinetics function for the UV-Vis, how to analyze data using Igor, and how to use the mixing attachment for the UV-Vis. We have also set up the tank which will hold our sea urchins when they arrive next semester. | ||
| − | == | + | ==Spring Semester 2019 Goals== |
| − | + | Moving forward, we hope to establish a final lab protocol for measuring peroxidase. The beginning of the semester will focus on adjusting the sea urchins to their tank and effectively monitoring their environment. We will be collaborating with Laura Sanchez to synthesize the sea urchin ovoperoxidase. | |
Latest revision as of 19:58, 30 June 2020
This research project involved the oxidation of guaiacol using the reaction between horseradish peroxidase and hydrogen peroxide. The purpose of this project was to develop a protocol for measuring the activity of a peroxidase using the UV-Vis.
Standard Mixing Procedure
The mixture of Peroxidase UV-Vis assays requires the use of a Guaiacol stock solution and a hydrogen peroxide stock solution.
The stock solutions are prepared as follows:
Guaiacol Stock Solution
49.5 microliters of Guaiacol is added to a 25 mL pH 8.0 buffer solution, which is comprised of 25 mL of DI water and a 8.0 pH buffer tablet that has been mixed thoroughly.
Hydrogen Peroxide Stock Solution
17 microliters of hydrogen peroxide is added to 1 mL of DI water.
Assay
5.0 mL Guaiacol stock solution is added to a vial.
10 microliters of the hydrogen peroxide stock solution is added to the Guaiacol stock solution.
The desired amount of peroxidase is then added to the assay before the UV-Vis is ran.
References
Purification and Assay publication
Progress as of December 2018
The main focus of the first semester of this research project has been to develop an efficient lab protocol for measuring the activity of a peroxidase. Since we will eventually be working with sea urchin peroxidase, background research was done on how to properly care for sea urchins as well as how to make UV-Vis assays for sea urchin peroxidase. With this information, we developed a standard mixing protocol for the UV-Vis assay using HRP. Several weeks have been spent modifying this procedure to produce more consistent results. Brad has taught me how to use the kinetics function for the UV-Vis, how to analyze data using Igor, and how to use the mixing attachment for the UV-Vis. We have also set up the tank which will hold our sea urchins when they arrive next semester.
Spring Semester 2019 Goals
Moving forward, we hope to establish a final lab protocol for measuring peroxidase. The beginning of the semester will focus on adjusting the sea urchins to their tank and effectively monitoring their environment. We will be collaborating with Laura Sanchez to synthesize the sea urchin ovoperoxidase.