Cong T. Trinh:AdhE enzyme assay on aldehydes
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.
