Such an approach is often the method of choice whenever an RNA—protein interaction or more complex assembly can be monitored in vitro. If specific binding is observed and one suspects that a specific protein is responsible and an antibody is available for that protein, it is possible to perform what is called supershift analysis.
Mobility shift approaches have been extremely valuable in deciphering the ordered pathway of assembly of large megadalton complexes such as the spliceosome.
In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel.
For large protein—RNA assemblies, such as pre-mRNA splicing complexes, it may be useful to try native agarose gels instead of polyacrylamide gels. Following nondenaturing gel electrophoresis, the labeled RNA is visualized by phosphorimaging or autoradiography, and the presence of shifted or not material is observed.
It is based on the observation that the electrophoretic mobility of a protein-nucleic acid complex is typically less than that of the free nucleic acid Fig. Although conceptually straightforward, it is important to note that gel composition and percentage as well as electrophoresis conditions can have profound effects on the results obtained.
By comparison with a set of standard dilutions of free probe run on the same gel, the number of moles of protein can be calculated. Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequence for the binding protein.
Competition between fluorophore- or biotin-labeled probe and unlabeled DNA of the same sequence can be used to determine whether the label alters binding affinity or stoichiometry. To distinguish between specific and nonspecific binding, competition experiments are performed.
In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. Here, the antibody is included in the initial binding incubation before electrophoresis.
Principle[ edit ] A mobility shift assay is electrophoretic separation of a protein—DNA or protein—RNA mixture on a polyacrylamide or agarose gel for a short period about 1. Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.
Methods used in performing the assay differ for each purpose, and a large number of variants have been described in the literature see Table 1.
Alternatively, the dye can be added to a free lane on the gel to monitor electrophoresis and prevent possible interference with protein binding.
The current, widely-used assay differs little from that originally described by Fried and Crothers 7 and Garner and Revzin 8although precursors to the technique can be found in the earlier literature 9 — Do not overload the wells. In this case, assembly can be monitored in the presence or absence of an energy source ATP or a specific RNP small nuclear ribonucleoprotein or auxiliary factor s.
Mix by pipetting up and down. A representative protocol is provided and commonly used variants are discussed. If the predicted consensus sequence fails to compete for binding, identification of the transcription factor may be aided by Multiplexed Competitor EMSA MC-EMSAwhereby large sets of consensus sequences are multiplexed in each reaction, and where one set competes for binding, the individual consensus sequences from this set are run in a further reaction.One important technique for studying gene regulation and determining protein–DNA interactions is the electrophoretic mobility shift assay (EMSA).
An advantage of studying protein–DNA interactions by an electrophoretic assay is the ability to resolve complexes of different stoichiometry or conformation. The electrophoretic mobility shift assay (EMSA), or gel mobility shift assay, is a popular and powerful technique for the detection of RNA–protein interactions.
It relies on the fact that naked RNA has certain mobility on nondenaturing gels, but if the RNA is bound by protein, the mobility of the RNA is reduced. The electrophoretic mobility shift assay (EMSA) is a rapid and sensitive method to detect protein-nucleic acid interactions 1 – 6.
It is based on the observation that the electrophoretic mobility of a protein-nucleic acid complex is typically less than that of the free nucleic acid (Fig.
1). An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein.
The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis.
By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.
The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents.
UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin.Download