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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 = 42.6
Average Z' factor at maximum response rate = 0.73
Average coefficient of variation (CV%) = 8.7%
Excellent signal to noise ratio
Cignal AP-1 reporter assay showed activation of MAPK signaling: 293-H cells were transfected with AP-1 reporter, negative control and positive control (for transfection protocol refer our user manual). After 24 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin) and cells were treated with 10 ng/ml of PMA or an equal volume of DMSO for 18 hours. Dual Luciferase assay was performed 42 hours after transfection, 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 AP-1 reporter assay measured 43 fold increase in the AP-1 transcription activity and, in turn, in corresponding MAPK signaling by 10 ng.ml PMA.
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