Methods to study transcription factor activation
Advantages and disadvantages of the most commonly used assays to study transcription factor activation.
Electrophoretic mobility shift assay (EMSA)/gel shift assay
The EMSA technique is the most popular technique to detect protein-DNA interactions. EMSA is based on the principle that protein-DNA complexes migrate slower than free linear DNA fragments in a non-denaturing gel electrophoresis.
- Advantages: simple to perform, high sensitivity, can also be used for studying RNA:protein interactions.
- Disadvantages: traditionally requires radioactive probes (although new biotin or fluorescent probes are now available), low-throughput, slow, non-quantitative.
Chromatin precipitation (ChIP)
ChIP is used to identify protein-DNA binding events in their natural, intracellular context. It is based on the detection of a specific protein located in a chromatin fraction that has been selectively enriched. The relative abundance of the protein at one or more locations in the genome can be determined using qPCR. It can be further used to identify the specific binding sequences in the genome (ChIP-seq).
- Advantages: works in vivo, shows interactions with other proteins, for studying transcription factor interaction changes over time.
- Disadvantages: requires specific ChIP-grade antibody to the transcription factor of interest, requires a lot of optimization.
ELISA-based assay
This assays detects the binding of transcription factors to DNA response elements bound to a microplate. It can be used to determine the effect of mutations or pharmaceutical compounds in the activation of the signal.
- Advantages: high-throughput, quick, (semi-)quantitative (can be quantitative if a standard curve is created with purified active protein).
- Disadvantages: requires specific antibody to the transcription factor of interest
If you need a quick and simple method to study activation of factors such as NFkB, FOXO1 or c-myc, our assays can give you the answer in less than four hours.
Nuclear extracts from K-562 cells stimulated with 5 µg TPA were assayed for activity of AP1 family members c-Jun, c-Fos, FosB, JunB, JunD and Fra-1 using AP1 (c-Fos/FosB/Fra1/c-Jun/JunD) Transcription Factor Assay Kit (Colorimetric) (ab207196). Extracts were incubate in the absence (grey) or presence of wild-type (black) or mutated (white) consensus binding oligonucleotides.