| Description |
The Cignal C/EBP Reporter Assay is designed
to monitor the activity of C/EBP-regulated signal transduction pathways in
cultured cells. The C/EBP, CCAAT/enhancer-binding protein, is a basic
leucine zipper transcription factor and is involved in the regulation of
mitotic growth arrest and differentiation. The C/EBP reporter is a mixture
of an inducible C/EBP responsive luciferase construct and a constitutively
expressing Renilla construct (40:1). The C/EBP-responsive luciferase
construct encodes the firefly luciferase reporter gene under the control
of a minimal (m)CMV promoter and tandem repeats of the C/EBP binding site.
We have experimentally optimized the number of response elements as well
as the intervening sequence between response elements to maximize the
signal to noise ratio. The constitutively expressing Renilla construct
encodes the Renilla luciferase reporter gene under the control of a CMV
immediately early enhancer/promoter and acts as an internal control for
normalizing transfection efficiencies and monitoring cell viability.
Using a simple dual-luciferase assay, you can easily monitor the activity
of C/EBP-mediated signaling pathways and determine the effect of various
treatments, such as gene knockdown, over-expression, and chemical
compounds on those pathways. For more information about the Cignal
Reporter Assays, please visit the Cignal
Reporter Assay home page.
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| Useful Links |
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| Related Products |
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| Materials Included / Packing List: |
| Please check the kit components immediately after you receive this package. SuperArray is only responsible for missing items reported within two (2) business days of receipt.
Kit Contents:
| Component |
Specification |
Concentration (total volume) |
| C/EBP Reporter |
A mixture of inducible C/EBP-responsive firefly
luciferase construct and constitutively expressing Renilla luciferase
construct (40:1). |
(100 ng/µl; 500 µl)* |
| Negative control |
A mixture of non-inducible firefly luciferase construct
and constitutively expressing Renilla luciferase construct
(40:1). |
(100 ng/µl; 500 µl) |
| Positive control |
A mixture of constitutively expressing GFP,
constitutively expressing firefly luciferase, and constitutively
expressing Renilla luciferase constructs (40:1:1). |
(100 ng/µl; 250 µl) |
* Supplied material provides sufficient reporter for 500 assays,
using recommended 96-well plate transfection protocol. The number
of assays per kit is a function of the assay plate format used
(refer to Cignal Reporter Assay User Manual).
Storage Conditions: The Cignal reporter assay
constructs are shipped ambient. Store all tubes at -20 ºC. |
| Brief Protocol: For Experienced
Users |
First time users, please refer to the complete
protocol in the Cignal Reporter Assays User Manual.
- Dilute transfection-ready reporter, negative control, and positive
control construct formulations.
- Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression
vector).
- Prepare appropriate combinations of reporter constructs, controls,
and test nucleic acids.
- Transfect plasmid mixtures separately into replicate wells of your
cell line of interest using an optimized transfection procedure for
the cell line under study.
- If applicable, 16 to 24 hours post-transfection, treat the
transfected cells with test proteins, peptides, or compounds of
interest.
- Two (2) to three (3) days post-transfection, assay the activities of
the signaling pathways under study, utilizing the dual luciferase
assay.
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| How It Works
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The Cignal Reporter Assays 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 impact of the experimental
treatments is determined by comparing the normalized luciferase activities
of the reporter to the identically treated negative control, across the
complete treatment regimen. 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.
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Performance Data
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General performance
Average maximum response rate = 10.3
Average Z' factor at maximum response rate = 0.71
Average coefficient of variation (CV%) = 7.2%
Excellent signal to noise ratio
Cignal C/EBP reporter assay can measure upregulation of C/EBP
transcription activity: 293-H cells were transfected with C/EBP 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 µg/ml streptomycin). After 24 hours of transfection,
cells were treated with 10mM LiCl for 18 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 C/EBP
reporter assay has shown 10 fold increase in C/EBP transcription activity
by 10 mM LiCl.
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