Team:Calgary/Notebook/Protocols/PrussianBlueFerritinMichaelisMentenKineticAnalysis

From 2013.igem.org

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<p>*Experiments conducted with TMB used stock hydrogen peroxide concentrations of 1%, while ABTS experiments used stock concentrations of 0.05%.
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<ol>
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<p> 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.
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<li>Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.</li>
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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>
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<li>Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.</li>
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<li>Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64μL)</li>
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<li> Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.</lio>
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</ol>
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<p>*Experiments conducted with TMB used a hydrogen peroxide concentration of 1%, while ABTS experiments used a hydrogen peroxide concentration of 0.05%.
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<h2>Substrate Variation</h2>
<h2>Substrate Variation</h2>
<h3>Reaction Mixture</h3>
<h3>Reaction Mixture</h3>
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<br>
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<p> 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.</p>
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<ol>
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<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

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

  1. Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
  2. Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
  3. Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
  4. Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64μL)
  5. 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

  1. Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
  2. Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
  3. Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
  4. Repeat the experiment multiple times for each substrate volume (0.5, 1, 2, 4, 6, 8 and 10μL)
  5. Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.
 

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