Related Experiment Videos
Detergents inhibit chloramphenicol acetyl transferase
1Section of Biochemistry Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
Biotechniques
|May 1, 1992
Summary
Detergents like Triton X-100 significantly inhibit chloramphenicol acetyl transferase (CAT) activity. Avoid using these detergents when preparing cell lysates for CAT reporter gene assays to ensure accurate results.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Chloramphenicol acetyl transferase (CAT) is a widely used reporter enzyme in transient expression studies.
- Accurate measurement of CAT activity is crucial for assessing gene expression levels.
- Cell lysis methods can impact enzyme activity, necessitating careful optimization.
Purpose of the Study:
- To investigate the effect of common detergents on chloramphenicol acetyl transferase (CAT) activity.
- To determine the suitability of different detergents for cell lysate preparation in CAT reporter assays.
Main Methods:
- Enzyme activity assays were performed on purified CAT.
- Various detergents, including Triton X-100, Nonidet P-40, sodium deoxycholate, sodium dodecyl sulfate, and Tween 20, were tested at different concentrations.
- CAT activity was measured spectrophotometrically.
Main Results:
- Triton X-100 and Nonidet P-40 demonstrated potent inhibition of CAT activity.
- Sodium deoxycholate and sodium dodecyl sulfate showed moderate inhibition.
- Tween 20 exhibited the least inhibitory effect on CAT activity.
Conclusions:
- Detergents, particularly non-ionic ones like Triton X-100 and Nonidet P-40, can significantly interfere with CAT enzyme activity.
- The use of these detergents during cell lysate preparation is not recommended when employing CAT as a reporter in transient expression experiments.
- Careful selection of lysis buffers is essential to maintain reporter enzyme integrity and ensure reliable experimental outcomes.