Team:Calgary/Notebook/Protocols/PrussianBlueFerritinMichaelisMentenKineticAnalysis

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<div id="Banner"><h1>Kinetic Analysis</h1></div>
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<h1>Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis</h1>

<h2>Hydrogen Peroxide Variation</h2>
<h3>Reaction Mixture</h3>
<ul>
<li> 10μL Prussian Blue Ferritin (22μg/mL) </li>
<li> 10μL Substrate* (TMB or ABTS, 10mg/mL) </li>
<li> 2-64μL Hydrogen Peroxide (1% or 0.05%)* </li>
<li> Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 220μL </li>
</ul>
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<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 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>
</ol>
<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>
<h3>Reaction Mixture</h3>
<ul>
<li> 10μL Prussian Blue Ferritin (22μg/mL) </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>
<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>
 


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