After the shift bands are observed in EMSA, super shift (supershift) or competitive EMSA (see in other section) may need for further investigating the shifted bands.
A. What is "Super shift (Supershift) EMSA"?
After the interaction of proteins and nucleic acids, an antibody is added to check if the antibody can bind to protein/DNA or protein/RNA complexes to create an even larger complex with a greater shift. This method is referred to as a super shift (supershift) EMSA, and bands with a greater shift are called supershifted bands.
B. Principle of Super Shift EMSA
The principle of Super Shift EMSA is easy understood. The binding of antibody to the protein (antigen) in the complexes of DNA/protein or RNA/protein would increase their size greatly and causes slow or stops mobility of complexes in the gel.
While there are characteristic shifts caused by specific protein(s) binding to target nucleic acid(s), a relative change in mobility does not identify the bound protein in a shifted complex. Identification of the protein bound to the probe is frequently accomplished by including an antibody that is specific for the putative DNA-binding in the binding reaction. If the protein of interest binds to the target DNA, the antibody will bind to that protein:DNA complex, further decreasing its mobility relative to unbound DNA in what is called a "supershift".
C. When is Super Shift EMSA needed?
After regular EMSA, Super Shift EMSA may need for (1) identifying the specificity of protein(s)/ nucleic acid(s) interaction, or (2) for determining the activated subunit of transcriptional factor family in the Protein/nucleic acid complexes.
Many transcriptional factors have many subunits to bind to a same domain in different cell lines or issues. For example, human NFkB has several subunits; p65, p52, p50, c-rel and v-rel. All these subunits are able to bind to the kB domain. As conventional EMSA cannot tell subunits involving in the protein/ nucleic acid complexes, supershift EMSA is a unique way to identify the components of protein/DNA complex in EMSA.
D. What antibodies can be used for Super Shift EMSA?
Not all of antibodies for a specific transcriptional factor can be used for Super Shift EMSA. The antibodies used with Super Shift EMSA should meet these standard: 1) the domain antibody recognized should be on the surface of protein-nuclear acid complexes, 2) the domain on the protein-nucleotide acid complexes should not be overlapped with interactive area of protein-nuclear acid complexes, 3) The antibodies used for Western blotting will not work with Supershift EMSA because antibody will only recognize the denatured prim structure of proteins. 4) The domain should be on the surface of the complexes, so that the antibody can access to bind. 5）Few antibodies made by injecting peptides can be used for supershift EMSA.
E. How to do Super Shift EMSA?
In order to get an expecting result from supershift EMSA, the experiment should consider following points:
(1) The cell or tissue extracts
should have activated proteins/factors enabled to bind to
DNA or RNA probes.
(2) Shifted bands of protein-nuclear acid complexes must be detected in conventional EMSA.
(3) Make sure the antibodies are enable to bind to native proteins in protein/nucleic acid complexes.
(4) Make sure the antibodies can be used for supershift EMSA.
(3) Antibodies should be added to reaction mixture after the reaction of protein and DNA/RNA.
(4) There should be no oxidants in reaction system.
(5) Nuclear proteins used for EMSA are isolated with a high-salt buffer. Higher volume of nuclear extracts may prevent the binding of antibody to protein/nucleic acid complexes.
(6) Usually, the concentration of antibodies used for supershiftEMSA is much higher than that used for other immunoassays such as Western-blotting or ELISA etc.
(7) A classic supershift EMSA includes these reactions: 1) A sample without activated target proteins (negative sample) + labeled probes, 2) A sample with activated target proteins + labeled probe (positive sample), 3) positive sample + labeled probe + low dose of specific antibody (supershift low), 4) positive sample + labeled probe + high dose of specific antibody (supershift high), 5) positive sample + labeled probe + low dose of non-specific antibody (unrelated low), 6) positive sample + labeled probe + low dose of non-specific antibody (unrelated high).
F. Explanation of Super Shift EMSA
A sample picture (picture 2) of supershift STATs EMSA from one of our publications (Blood 1999, 93:2369-79), in which supershifted bands can be seen clearly. Depended on types of antibodies, supershifted band(s) may not be observed in EMSA gels (picture 3 from another article of ours, JBC 1999,274:13877–85), presenting another type of positive supershift EMSA when polyclonal antibody, which causes; 1) no supershift bands, 2) disappeared or weaker protein-nuclear acid complexes than that of controls, and 3) aggregates in the loading wells.
Positive supershift EMSA can be judged by one of follows; 1) Specific shifted bands become weaker or disappearing, but non-specific bands are not affected, with a clear supershifted bands of antibody-protein-nuclear acid complexes when monoclonal or peptides-immunized antibodies are used. 2) Specific shifted bands become weaker or disappearing, but non-specific bands are not affected, without supershifted bands of antibody- protein-nuclear acid complexes in the gel when polyclonal antibodies are used. 3) In all the cases, if non-specific bands are also disappeared or become weaker, indicating an amount of antibodies used is too much and the experiments need to repeat by adjusting antibody usage.