Difference between revisions of "Team:UiOslo Norway/Experiments/In vitro Assay Methanol Dehydrogenase"

 
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   <li><p>Incubate at 37 °C for 3 hours</p></li>
 
   <li><p>Incubate at 37 °C for 3 hours</p></li>
 
   <li><p>Harvest the culture by centrifugation at 8000 x g for 5 minutes</p></li>
 
   <li><p>Harvest the culture by centrifugation at 8000 x g for 5 minutes</p></li>
−
   <li><p>Resuspend the cell pellet in 1 ml 50 mM K2HPO4 buffer (pH 7.4)</p></li>
+
   <li><p>Resuspend the cell pellet in 1 ml 50 mM K<sub>2</sub>HPO<sub>4</sub> buffer (pH 7.4)</p></li>
 
   <li><p>Add glass beads (0.1mm diameter) and disrupt the cells by shaking for 12 minutes</p></li>
 
   <li><p>Add glass beads (0.1mm diameter) and disrupt the cells by shaking for 12 minutes</p></li>
 
   <li><p>Centrifugation at 16000 x g for 5 minutes</p></li>
 
   <li><p>Centrifugation at 16000 x g for 5 minutes</p></li>
 
   <li><p>Keep the soluble fraction for the assay</p></li>
 
   <li><p>Keep the soluble fraction for the assay</p></li>
 
   <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
 
   <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
−
   <li><p>Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4</p></li>
+
   <li><p>Add preheated 50 mM K<sub>2</sub>HPO<sub>4</sub> buffer (pH 7.4) containing 5 mM MgSO<sub>4</sub></p></li>
 
   <li><p>Add 50 µl of soluble fraction</p></li>
 
   <li><p>Add 50 µl of soluble fraction</p></li>
 
   <li><p>Add 1 M methanol</p></li>
 
   <li><p>Add 1 M methanol</p></li>
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<p>
 
<p>
 
<ul>
 
<ul>
−
  <li><p> </p></li>
 
 
   <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
 
   <li><p>Perform the enzyme assay in a total volume of 1 ml</p></li>
−
   <li><p>Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4</p></li>
+
   <li><p>Add preheated 50 mM K<sub>2</sub>HPO<sub>4</sub> buffer (pH 7.4) containing 5 mM MgSO<sub>4</sub></p></li>
−
   <li><p>Add 50 ul of soluble fraction</p></li>
+
   <li><p>Add 100 µg of purified MEDH2 </p></li>
−
   <li><p>Add 1 M methanol</p></li>
+
   <li><p>Add 0.5 M methanol</p></li>
−
   <li><p>Add 500 uM NAD+</p></li>
+
   <li><p>Add 0.5 mM NAD+</p></li>
 
   <li><p>Detect the absorption at 340 nm</p></li>
 
   <li><p>Detect the absorption at 340 nm</p></li>
 
</ul>
 
</ul>
  
 +
<div class="clear"></div>
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<center><h1>iGEM UiOslo 2015 is sponsored by:</h1>
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Latest revision as of 12:20, 18 September 2015

In vitro Methanol Dehydrogenase assay using E. coli raw extract

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  • Inoculate a preculture LB media containing the appropriate antibiotic and incubate at 37 °C overnight shaking at 220 rpm.

  • Inoculate a mainculture (50 ml) and induce protein expression by adding IPTG to a final concentration of 1 mM at an OD600 of 0.4- 1.0

  • Incubate at 37 °C for 3 hours

  • Harvest the culture by centrifugation at 8000 x g for 5 minutes

  • Resuspend the cell pellet in 1 ml 50 mM K2HPO4 buffer (pH 7.4)

  • Add glass beads (0.1mm diameter) and disrupt the cells by shaking for 12 minutes

  • Centrifugation at 16000 x g for 5 minutes

  • Keep the soluble fraction for the assay

  • Perform the enzyme assay in a total volume of 1 ml

  • Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4

  • Add 50 µl of soluble fraction

  • Add 1 M methanol

  • Add 200 µM NAD+

  • Detect the absorption at 340 nm

In vitro Methanol Dehydrogenase assay using purified NAD+ dependent Methanol Dehydrogenase

Back to Protocols


  • Perform the enzyme assay in a total volume of 1 ml

  • Add preheated 50 mM K2HPO4 buffer (pH 7.4) containing 5 mM MgSO4

  • Add 100 µg of purified MEDH2

  • Add 0.5 M methanol

  • Add 0.5 mM NAD+

  • Detect the absorption at 340 nm

iGEM UiOslo 2015 is sponsored by: