The following article scrutinizes the assays most frequently used in the study of activation of transcription factors, listing their relative advantages and disadvantages.
Electrophoretic Mobility Shift Assay (EMSA)/Gel Shift Assay
Of all the techniques to detect protein-DNA interactions, the EMSA method is the most popular.
This technique is founded on the principle that in non-denaturing gel electrophoresis, protein-DNA complexes migrate more slowly than free linear DNA fragments.
One of the main advantages of this technique is its simplicity to perform. It also has high sensitivity and can be used for analyzing RNA:protein interactions.
One disadvantage is that this method normally requires radioactive probes (however, this requirement can be prevented by the use of newly available biotin or fluorescent probes). This technique is also slow, non-quantitative and has a low-throughput.
Chromatin Precipitation (ChIP)
To identify protein-DNA binding events in their natural, intracellular context, ChIP can be used.
This technique detects specific proteins located in a selectively enriched chromatin fraction.
The relative abundance of these proteins can be determined at one or more locations in the genome with the use of qPCR. Furthermore, qPCR can be used to identify the genome’s specific binding sequences (ChIP-seq).
The advantages of ChIP are that it works in vivo and shows interactions with other proteins. It can also be used to study transcription factor interaction alterations over time.
However, this method requires a great deal of optimization and needs a specific ChIP-grade antibody tailored to the transcription factor of interest.
The ELISA-based assay can detect transcription factors’ binding to DNA response elements bound to a microplate. It can also establish the impact of pharmaceutical compounds or mutations in the signal’s activation.
The advantages of this assay include its speed and high-throughput. It is also (semi-)quantitative (can be quantitative if a standard curve is created with purified active protein).
A disadvantage of this method is that it requires specific antibody to the transcription factor of interest.
Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.
Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.