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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 = 191.5
Average Z' factor at maximum response rate = 0.93
Average coefficient of variation (CV%) = 2.4%
Excellent signal to noise ratio
Excellent signal to noise ratio
Cignal NFκB reporter assay showed dose dependent upregulation of NFκB signaling activity: 293-H cells were transfected with NFκB 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 + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 ?/ml streptomycin). After 24 hours of transfection, cells were treated with different doses of recombinant human tumor necrosis factor alpha (hTNFα) protein for another 24 hours. Dual Luciferase assay was performed 48 hours after the 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 NFκB reporter assay measured 191 fold increase in NFκB transcription activity by 50 ng/ml hTNFα.
Cignal NFκB reporter assay can measure down regulation of NFκB signaling activity by RelA shRNA or NFκB siRNA: 293 H cells were transfected with NFκB reporter construct, negative control construct and positive control construct along with NFκB1siRNA, RelA shRNA, negative control siRNA and negative control shRNA (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to complete. Dual Luciferase assay was performed 72 hours after the 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.
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