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Assay for Rab geranylgeranyltransferase using size exclusion chromatography
V S Hung1, P Low, E Swiezewska
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, Warsaw, 02-106, Poland.
Analytical Biochemistry
|February 13, 2001
Summary
A new chromatographic assay simplifies Rab geranylgeranyltransferase (Rab GGTase) activity measurement. Adding 2-propanol improves assay reliability by reducing noncovalent complexes, enabling routine analysis of prenyltransferase activity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Rab geranylgeranyltransferase (Rab GGTase) is crucial for protein prenylation.
- Accurate and reliable assays are needed to study Rab GGTase activity.
- Existing methods may face challenges with reproducibility due to noncovalent complexes.
Purpose of the Study:
- To develop a simple and reliable chromatographic assay for Rab GGTase.
- To improve the reproducibility and accuracy of Rab GGTase activity measurements.
- To demonstrate the assay's applicability across different biological samples.
Main Methods:
- Development of a chromatographic assay using Sephadex G-25 superfine minicolumns.
- Inclusion of 2-propanol in the assay to minimize noncovalent lipid-protein complex formation.
- Measurement of Rab prenyltransferase activity in various plant and animal samples.
Main Results:
- The developed assay effectively separates reaction components.
- Addition of 2-propanol reduced noncovalent complex formation by approximately 90%.
- The assay demonstrated high reproducibility and reliability for Rab GGTase activity determination.
Conclusions:
- A simple, reproducible, and reliable chromatographic assay for Rab GGTase has been established.
- The method is suitable for routine evaluation of Rab prenyltransferase activity.
- This assay can be applied to diverse biological sources, including wheat seedlings and rat brain cytosol.