Emsa-based Method
Emsa-based Method - Related Videos
06:57
Electrophoretic Mobility Shift Assay (EMSA)
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2023
The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation. This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video...
09:57
siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts
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Cited by 3 •
2016
This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.
11:35
Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
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Cited by 15 •
2016
We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.
07:51
Chromatography-based Biomolecule Purification Methods
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2023
In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase,...
12:44
Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
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Cited by 37 •
2014
Here we present a protocol to analyze RNA/protein interactions. 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.
FAQs
The shift reflects a change in electrophoretic mobility after a protein associates with the probe. Under native gel conditions, the protein–nucleic-acid complex remains sufficiently intact to migrate differently from free labeled DNA or RNA, allowing the two forms to appear as separate bands. This comparison provides evidence of binding rather than merely protein presence.
Competition assays test whether the observed interaction depends on a particular nucleic-acid sequence. A competing nucleic acid is introduced alongside the labeled probe; reduced shifted signal indicates that binding can be challenged by that competitor. This approach helps distinguish sequence-specific recognition from an interaction that occurs broadly with nucleic acids.
Supershift assays add an antibody to the binding reaction to help identify the protein responsible for a shifted complex. If the antibody associates with that complex, its added mass can alter the migration pattern, producing a further shift. In gene-regulation studies, this evidence connects a mobility change with a candidate transcription factor rather than leaving the protein identity unresolved.
Probe choice determines which nucleic-acid interaction the assay examines. A labeled DNA probe can represent a regulatory region such as a promoter, whereas labeled RNA allows analysis of protein–RNA binding. Comparing the migration of free and protein-associated probe then links the detected shift to the selected nucleic-acid target and its biological question.
An EMSA-based workflow begins by combining a purified or cellular protein preparation with a labeled DNA or RNA probe. After incubation, the mixture is loaded onto a native gel so complexes can remain associated during separation. The resulting pattern is interpreted by locating slower-migrating probe-containing material relative to unbound probe.
Within biology, these assays are useful for examining transcription-factor activity, promoter recognition, and gene regulation. They can also connect altered protein–nucleic-acid binding with molecular mechanisms underlying cellular responses. Because competition tests specificity and supershifts support protein identification, the method can provide both interaction evidence and context for interpreting regulatory mechanisms.