326
Related Techniques
following is a method based on the use of the Authenti kit (Corning),
which is a kit developed especially for isozyme analysis.
Materials
Equipment - Authenti kit agarose gel electrophoresis apparatus
- Incubator (37°C)
- Centrifuge and centrifuge tubes
- Sonic cell disintegrator
- Pasteur pipettes
- 5-ml pipette
Solutions - Authenti kit extraction buffer (50 mM Tris-HCI, pH 7.5, 1 mM EDTA,
2% Triton X-I00; store the extraction buffer at 4°C)
- Authenti kit enzyme stabilization buffer
- Electrophoresis buffer (0.05 M PHAB)
- Buffer for enzyme activity measurements (0.1 M PHAB)
- Enzyme substrate reagent
Dissolve one vial of Authenti kit enzyme substrate powder in 0.5 ml
of 0.1 M PHAB (use within 30 min); usually substrates for isocitrate
dehydrogenase, malic enzyme, phosphoglucose isomerase, and phosphoglucomutase are used.
Miscellaneous - Authenti kit film (0.8% agarose gel)
- Filter paper
Preparation
of enzyme
sample
Electrophoresis
Procedure
1. Collect the cells to be analysed by dispersing the cells and by centrifuging (150 g for 5 min) the resulting cell suspension. The number of
cells needed depends on the size of cells. Harvesting between 5 x 10 6
and 1 x 10 7 cells is recommended.
2. Wash the cells with physiological saline two to three times by centrifugation at 150 g for 5 min.
3. Suspend the cells in 0.1 ml Authenti kit extraction buffer.
4. Sonicate the cells (or cells may be disintegrated by freeze-thaw treatment of the cells), and centrifuge the cells at 2,000 g for 10 min.
5. Add approximately the same volume of Authenti kit enzyme stabilization buffer, and keep the sample frozen at below -30°C until electrophoresis.
1. After thawing of the frozen sample, apply 1 ~ sample to the Authenti
kit film.
2. Set the film on the Authenti kit agarose gel electrophoresis apparatus.
3. Run electrophoresis at 160 V for 25 min.
Related Techniques
following is a method based on the use of the Authenti kit (Corning),
which is a kit developed especially for isozyme analysis.
Materials
Equipment - Authenti kit agarose gel electrophoresis apparatus
- Incubator (37°C)
- Centrifuge and centrifuge tubes
- Sonic cell disintegrator
- Pasteur pipettes
- 5-ml pipette
Solutions - Authenti kit extraction buffer (50 mM Tris-HCI, pH 7.5, 1 mM EDTA,
2% Triton X-I00; store the extraction buffer at 4°C)
- Authenti kit enzyme stabilization buffer
- Electrophoresis buffer (0.05 M PHAB)
- Buffer for enzyme activity measurements (0.1 M PHAB)
- Enzyme substrate reagent
Dissolve one vial of Authenti kit enzyme substrate powder in 0.5 ml
of 0.1 M PHAB (use within 30 min); usually substrates for isocitrate
dehydrogenase, malic enzyme, phosphoglucose isomerase, and phosphoglucomutase are used.
Miscellaneous - Authenti kit film (0.8% agarose gel)
- Filter paper
Preparation
of enzyme
sample
Electrophoresis
Procedure
1. Collect the cells to be analysed by dispersing the cells and by centrifuging (150 g for 5 min) the resulting cell suspension. The number of
cells needed depends on the size of cells. Harvesting between 5 x 10 6
and 1 x 10 7 cells is recommended.
2. Wash the cells with physiological saline two to three times by centrifugation at 150 g for 5 min.
3. Suspend the cells in 0.1 ml Authenti kit extraction buffer.
4. Sonicate the cells (or cells may be disintegrated by freeze-thaw treatment of the cells), and centrifuge the cells at 2,000 g for 10 min.
5. Add approximately the same volume of Authenti kit enzyme stabilization buffer, and keep the sample frozen at below -30°C until electrophoresis.
1. After thawing of the frozen sample, apply 1 ~ sample to the Authenti
kit film.
2. Set the film on the Authenti kit agarose gel electrophoresis apparatus.
3. Run electrophoresis at 160 V for 25 min.