Transcription Factor Activity Assay Sample Protocol
Transcription factor activity assays
DNA-binding ELISA kits to measure active transcription factors - NF-kB, p53, CREB, STAT3, SMAD, c-Jun, MYC, GATA and 200+ more, including phospho-specific assays. A fast, safe, high-throughput alternative to EMSA.
Why use a DNA-binding ELISA?
Assay transcription-factor activity without radioactivity or gels.
- Simple - measure transcription-factor DNA binding across a 96-well plate at once.
- Rapid - far quicker than EMSA, western blot or gel-shift methods.
- Safe - no radioactive labelling required.
- High throughput - run many samples simultaneously in 96-well format.
- Choice - total and phospho-specific transcription-factor assays available.
Applications
- Semi-quantitatively detect transcription factor-DNA complexes in nuclear and cell lysates.
- Analyse signal-transduction pathways.
- Investigate target candidates in drug development.
- Screen for inhibitors and activators of transcription-factor activity.
- Establish the effects of phosphorylation on transcription-factor activation.
Browse transcription factor assays
Filter by total or phospho-specific, or search for your transcription factor.
How the assay works
The Assay Genie Transcription Factor Activity Assay is an indirect ELISA for detecting active transcription factors in eukaryotic nuclear or cell lysates. Streptavidin is bound to the plate, and biotinylated double-stranded (dsDNA) oligonucleotides bind to it via the high-affinity biotin-streptavidin interaction.
After blocking, the sample is added and active transcription factors bind their dsDNA response element. A primary antibody recognises the bound transcription factor, and an HRP-conjugated secondary antibody enables colorimetric detection with TMB substrate.
The peroxidase reaction produces a blue product proportional to the amount of target; adding stop solution (2 N sulfuric acid) turns it yellow, and absorbance is read at 450 nm to determine target activity in the sample.
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Transcription factor assay FAQs
What can a transcription factor activity assay measure?
It semi-quantitatively detects active (DNA-bound) transcription factors in nuclear or whole-cell lysates - useful for studying signalling, screening activators/inhibitors, and assessing the effect of phosphorylation on activation.
How is this different from EMSA or western blot?
This DNA-binding ELISA is faster, non-radioactive and higher-throughput than EMSA/gel-shift or western blotting, letting you assay many samples in a 96-well plate simultaneously.
Do you offer phospho-specific assays?
Yes - alongside total transcription-factor assays there are many phospho-specific assays (e.g. NF-kB p65, p53, CREB, STAT1, c-Jun at defined phospho-sites) to study activation state.
My kit was left at room temperature - can I still use it?
It is most likely still usable, especially after only a short time, though activity may diminish. The most temperature-sensitive components are the standards, HRP-streptavidin and detection antibody.
What sample types are used?
Eukaryotic nuclear extracts or whole-cell lysates. Correct lysate preparation and storage is important for a strong, specific signal.
Can't find your transcription factor?
We offer 180+ targets and custom assay development - our scientific team can help you find or build the right assay.