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Team:Calgary/Notebook/Protocols/PrussianBlueFerritinMichaelisMentenKineticAnalysis
From 2013.igem.org
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<h1>Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis</h1> | <h1>Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis</h1> | ||
| - | < | + | <h2>Hydrogen Peroxide Variation</h2> |
| - | < | + | <h3>Reaction Mixture</h3> |
<ul> | <ul> | ||
<li> 10μL Prussian Blue Ferritin (<b>CONCENTARION</b>) </li> | <li> 10μL Prussian Blue Ferritin (<b>CONCENTARION</b>) </li> | ||
| Line 20: | Line 20: | ||
<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. | <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. | ||
</p> | </p> | ||
| + | <h2>Substrate Variation</h2> | ||
| + | <h3>Reaction Mixture</h3> | ||
| + | <ul> | ||
| + | <li> 10μL Prussian Blue Ferritin (<b>CONCENTARION</b>) </li> | ||
| + | <li> 0.5-10μL Substrate (TMB or ABTS, 10mg/mL) </li> | ||
| + | <li> 32μL Hydrogen Peroxide (30%) </li> | ||
| + | <li> Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 242μL </li> | ||
| + | </ul> | ||
| + | <br> | ||
| + | <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> | ||
| + | |||
| - | |||
</section> | </section> | ||
</html> | </html> | ||
Revision as of 17:18, 26 September 2013
Kinetic Analysis
Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis
Hydrogen Peroxide Variation
Reaction Mixture
- 10μL Prussian Blue Ferritin (CONCENTARION)
- 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
*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 (CONCENTARION)
- 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.
