Team:Calgary/Notebook/Protocols/PrussianBlueFerritinMichaelisMentenKineticAnalysis
From 2013.igem.org
(Difference between revisions)
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<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 | + | <li>Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64μL)</li> |
<li> Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.</lio> | <li> Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.</lio> | ||
</ol> | </ol> | ||
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</ul> | </ul> | ||
<br> | <br> | ||
- | < | + | <ol> |
+ | <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>Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.</li> | ||
+ | <li>Repeat the experiment multiple times for each substrate volume (0.5, 1, 2, 4, 6, 8 and 10μL)</li> | ||
+ | <li> Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.</lio> | ||
+ | </ol> | ||
Latest revision as of 00:44, 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 64μL)
- Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.
*Experiments conducted with TMB used a hydrogen peroxide concentration of 1%, while ABTS experiments used a hydrogen peroxide concentration of 0.05%.
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
- 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 substrate volume (0.5, 1, 2, 4, 6, 8 and 10μL)
- Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.