Overview of our system

Vector information

Protocols

Protoplast isolation

Gene transfection

Luciferase assay

Western blotting

Results

Transfect efficiency

Gene expression levels of dual expression vectors

Protein interaction

Acknowledgements

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Luminescence detections

Materials

• Luminometers
• Veritas^TM microplate Luminometer (Turner BioSystems, CA)
• VICTOR³ Multilable Plate Reader (PerkinElmer Life and Analytical Sciences, CT).

For Renilla luciferase assays

ViviRen™ Live Cell substrate (Promega, Cat.#E6492)

Stock solution (6 mM solution)

• ViviRen™ is dissolved in DMSO (dimethyl sulfoxide) as a 6 mM solution to store at -80 °C.

Working solution (60 μM solution)

• The stock solution is diluted 100 times with W5 buffer before each assay.

For the firefly luciferase assays

Beetle luciferin (Promega, Cat.#E1603)

• Stock solution
  • Beetle luciferin (Beetle Luciferin, potassium salt) is dissolved in sterile water to a final concentration of 7.8 mM to store at -80 °C.
• Working solution (780 μM solution)
  • The stock solutions is diluted 10 times with the W5 buffer before each assay.

Protocol

1. 10 μl of working solution are added to each well of a 96-well plate
2. Incubate for at least 15 min at room temperature in the dark before measuring the luminescence.
3. The luminescence in each well is quantified with 10 second integration periods using luminometers.

Result

Protein-protein interaction of histone proteins

 

Last Update 8/1/07