Lane 1 is a negative control, and contains only DNA. Lane 2 contains protein as well as a DNA fragment that, based on its sequence, does not interact. Lane 3 contains protein and a DNA fragment that does react; the resulting complex is larger, heavier, and slower-moving. This situation is contrived, and in reality there would likely be a second band in lane 3 owing to dissociation of the DNA-protein complex.
EMSA stands for electrophoretic mobility shift assay. Mobility shift assays are also known as gel shift assay, band shift assay or gel retardation assay.
EMSA is a common technique used to study DNA-protein or RNA-protein interactions.
Function of EMSA Procedure
EMSA function to analyze the set of trans-acting proteins (or protein) that interact with a specific cis-actingDNA or RNA sequences. There are in fact two types of EMSAs, including denaturing EMSA and non-denaturing EMSA.
Denaturing EMSA
Denaturing EMSA is conducted under denaturing conditions and functions to determine the number and size of the proteins that are directly binding to and interacting with a DNA or RNA nucleic acid sequence fragment. Denaturing EMSAs are done by UV-crosslinking any directly interacting proteins to the DNA or RNA fragment (by making covalent bonds), and then running the complex under denaturing conditions which removes any non-covalent interactions (such as protein-protein interactions).
Thus, you theoretically should result in nucleic acid fragments that are bound only to directly interacting protein factors.
Non-denaturing EMSA
Non-denaturing EMSA functions to determine the different types of complexes that bind to a DNA or RNA nucleic acid sequence fragment. Non-denaturing EMSAs are conducted under non-denaturing conditions.
and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation.
Principle of the Mobility Shift Assay
A mobility shift assay generally involves electrophoretic separation of a protein-DNA mixture on a polyacrylamide or agarose gel for a short period. The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape (see Gel electrophoresis). The control lane without protein present will contain a single band corresponding to the unbound DNA fragment. However, assuming the protein is capable of binding to the DNA fragment, the lane with protein present will contain another band that represents the larger complex of DNA bound to protein. From the ratio of bound to unbound DNA, the affinity of the protein to the DNA sequence may be determined.
Often, an extra lane is run with a competitor oligonucleotide to determine the most favorable binding sequence for the binding protein. The use of different oligonucleotides of defined sequence allows the identification of the precise binding site by competition. (Not shown in diagram). Variants of the competition assay are useful for measuring the specificity of binding and for measurement of association and dissociation kinetics.
For visualization purposes, the DNA fragment is usually labeled with a radioactive or fluorescent label, as standard ethidium bromide staining lacks the sensitivity to detect the relatively small amounts of DNA used in these experiments.
DNA EMSA Protocol
The DNA EMSA assay allows the analysis of the binding of proteins to DNA fragments. The premise of the assay is that DNA bound to protein will migrate slower (will be retarded in migration) in a gel matrix than unbound DNA during electrophoresis.
Most EMSA procedures user 32P labelling of the DNA fragment. However, it is also possible to detect the DNA via non-radioactive detection methods (see non-radioactive EMSA) (including fluorescence and biotin-streptavidin enyzme detection methods). Ethidium bromide staining is generally not sensitive enough since usually small amounts of DNA are used in this assay.
Key Features of Biotin-based DNA EMSA kits include the fact that they are highly sensitive HRP-based detection systems, which are comparable to 32P DNA binding assays. They are also safe, as no radioactivity is required.
