"
Team:Calgary/Notebook/Protocols/PrussianBlueFerritinMichaelisMentenKineticAnalysis
From 2013.igem.org
| Line 16: | Line 16: | ||
</ul> | </ul> | ||
<br> | <br> | ||
| - | |||
| - | |||
| - | < | + | <ol> |
<li>Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.</li> | <li>Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.</li> | ||
<li>Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.</li> | <li>Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.</li> | ||
<li>Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.</li> | <li>Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.</li> | ||
<li>Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64)</li> | <li>Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64)</li> | ||
| - | <li></ | + | <li> Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.</lio> |
| + | </ol> | ||
| + | <p>*Experiments conducted with TMB used stock hydrogen peroxide concentrations of 1%, while ABTS experiments used stock concentrations of 0.05%. | ||
| + | </p> | ||
<li></li> | <li></li> | ||
<li></li> | <li></li> | ||
Revision as of 00:35, 28 September 2013
Kinetic Analysis
Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis
Hydrogen Peroxide Variation
Reaction Mixture
- 10μL Prussian Blue Ferritin (22μg/mL)
- 10μL Substrate* (TMB or ABTS, 10mg/mL)
- 2-64μL Hydrogen Peroxide (1% or 0.05%)*
- Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 220μL
- Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
- Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
- Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
- Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64)
- Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.
*Experiments conducted with TMB used stock hydrogen peroxide concentrations of 1%, while ABTS experiments used stock concentrations of 0.05%.
The reaction was conducted in room temperature for 10 minutes and the absorbance value was recorded at 10 second intervals. Absorbance for TMB was taken at 650nm, and absorbance for ABTS was taken at 415nm. Eight replicates were conducted. Substrates were the last reagent to be added to the mix. The experiment was conducted multiple times for the addition of 2, 4, 6, 8, 12, 16, 32, and 64μL of hydrogen peroxide. Resulting slopes of these experiments were then used to generate a Michaelis-Menten plot.
Substrate Variation
Reaction Mixture
- 10μL Prussian Blue Ferritin (22μg/mL)
- 0.5-10μL Substrate (TMB or ABTS, 10mg/mL)
- 32μL Hydrogen Peroxide (30%)
- Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 242μL
The reaction was conducted in room temperature for 10 minutes and the absorbance value was recorded at 10 second intervals. Absorbance for TMB was taken at 650nm, and absorbance for ABTS was taken at 415nm. Eight replicates were conducted. Substrates were the last reagent to be added to the mix. The experiment was conducted multiple times for the addition of 0.5, 1, 2, 4, 6, 8, and 10μL of substrate. Resulting slopes of these experiments were then used to generate a Michaelis-Menten plot.
