Cong T. Trinh:AdhE enzyme assay on aldehydes

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Contents

AdhE enzyme assay on aldehydes

Materials and methods

50 mM MOPS, pH7. This buffer is used for assaying the His-tagged AdhE. 10 mM NADH stock (in MOPS, pH7). This chemical is quite stable when stored at -20°C. Thawing cycles does not affect the absorbance. 500 mM aldehyde (acetaldehyde, isobutyraldehyde, butyraldehyde). Enzyme. For example, enzyme concentration is 2.5 mg/mL, which is measured by Bradford method. Working concentration is 10 μg/mL. Note: In some instance, Fe(NH4)2 SO4 is added in MOPS with a working concentration of 0.03 mM. At this working concentration, the activity of acetaldehyde increases while the activities of butyraldehyde and isobutyraldehyde decrease.


Procedures

In 1 mL reaction, add the following components in order
i. 960 μL MOPS
ii. 10 μL NADH
iii. 4 μL of enzyme
iv. 10 μL aldehyde
Measure absorbance at 340 nm every 10 second for 180-360 seconds. The reaction is carried out at 37oC by using the circulating water.


Controls

Two types of the control experiment are carried out.

The first control experiment contains:

990 μL MOPS
10 μL NADH.

The second control experiment contains:

986 μL MOPS
10 μL NADH
4 μL enzyme.



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