| Cell Line Species/Tissue: |
Human
/
Kidney Epithelial |
| Transfection Reagent: |
PolyFect |
Nucleic Acid:
|
DNA |
Growth Medium:
|
|
Percent Serum (%):
|
|
Reporter System:
|
|
Plasmid Purification Method:
|
|
Plate Format:
|
35mm tissue culture dishes |
Number of Cells:
|
5x10e5 |
Percent Confluence(%):
|
|
Amount of Nucleic Acid (µg):
|
2 mg |
Amount of Enhancer (µl):
|
|
Amount of Reagent (µl):
|
no information |
Complex Incubation on Cells (hrs):
|
|
Analysis Performed Post-Transfection (hrs):
|
36 h |
Transfection Efficiency (%):
|
|
| |
| Any modifications to the protocol?:
|
| |
| Notes: |
| Human 293T cells (5´105) were seeded into 35 mm tissue culture
dishes and transiently transfected with 2 mg pG5E1b-luciferase
reporter and 2 mg plasmids expressing Gal4 DB domain or its fusion
proteins using PolyFect Transfection Reagent (QIAGEN). A 0.5 mg CMV-
b-Gal plasmid was co-transfected as an internal control. Cells were
harvested after 36 hours and lysed in 1% Triton X-100, 100 mM Tris
(pH 7.8), 2 mM EDTA, 2 mM DTT, 2 mg/ml Aprotinin, 2 mg /ml
Leupeptin, 2 mg /ml Pepstatin and 100 mg/ml PMSF. Luciferase
activity was determined on MonolightTM 3010 (PharMingen).
|
| References: |
| Pygopus encodes a nuclear protein essential for wingless/Wnt signaling
Belenkaya, T.Y., Han, C., Standley, H.J., Lin, X., Houston, D.W., Heasman, J. and Lin, X.
Development (2002) 129(17), 4089-101
|