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The Cignal Reporter Assays (luc) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The change in the activity of each signaling pathway is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.
General performance
Average maximum response rate = 26.8
Average Z' factor at maximum response rate = 0.73
Average coefficient of variation (CV%) = 6.5%
Excellent signal to noise ratio
Cignal E2F reporter assay showed activation of E2F
cell cycle pathway: 293-H cells were transfected with E2F
reporter, negative control and positive control (for transfection protocol
refer our user manual). After 16 hours of transfection, medium was changed
to assay medium (Opti-MEM + 0.5% FBS + 0.1 mM NEAA + 1mM Sodium pyruvate +
100 U/ml penicillin + 100 ug/ml streptomycin). After 40 hours of
transfection, cells were treated with 10% serum, and 10% serum and 100 ng/ml
EGF for 6 hours. Dual Luciferase assay was performed, and promoter
activity values are expressed as arbitrary units using a Renilla reporter
for internal normalization. Experiments were done in triplicates, and the
standard deviation is indicated. Cignal E2F reporter assay measured 27
fold increase in E2F transcription activity in response to serum.
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