Team:UiOslo Norway/Experiments/In vitro Assay Methanol Dehydrogenase

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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

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• 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 ul of soluble fraction

• Add 1 M methanol

• Add 500 uM NAD+

• Detect the absorption at 340 nm