Thermo Scientific Pierce Protein Interaction Technical Handbook

Electrophoretic-Mobility Shift Assays (EMSA)
EMSA Applications
The Supershift Reaction
This technique can aid in the identification of the DNA-bound
protein. This is accomplished by including an antibody, specific
for the DNA-binding protein, to 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.” In addition to
antibodies, supershift reactions could include other secondary
or indirectly bound proteins.
Lane 1 2 3 4
EBNA Extract _ + + +
Unlabeled EBNA DNA _ _ + +
Unlabeled Oct-1 DNA _ _ _ +
DNA + EBNA
Protein
Shift-Western Blot
This application involves transferring the resolved protein:DNA
complexes to stacked nitrocellulose and anion-exchange
membranes. Proteins captured on the nitrocellulose membrane
can be probed with a specific antibody (Western blot) while
autoradiography or chemiluminescent techniques can detect the
DNA on the anion-exchange membrane.
Alternatively, DNA can be labeled with a biotinylated or haptenlabeled
dNTP, then probed and detected using an appropriately
sensitive fluorescent or chemiluminescent substrate. We offer
a chemiluminescent EMSA system (Product # 20148) and a kit to
facilitate labeling DNA with biotin (Product # 89818). The EMSA
Kit offer detection levels rivaling that of isotopic-based systems.
Free DNA
Figure 12. EMSA results using the EBNA control system. Biotin-labeled
60 bp duplex bearing the EBNA-1 binding sequence was incubated with an
extract in which the EBNA-1 protein was overexpressed. The binding buffer
was supplemented with 50 ng/µl poly(dI•dC), 10% glycerol and 0.05% NP-40.
Exposure time was 30 seconds with X-ray film.
Nonspecific Competitor
Nonspecific competitor DNA such as poly(dI•dC) or poly(dA•dT)
is included in the binding reaction to minimize the binding of
nonspecific proteins to the labeled target DNA. These repetitive
polymers provide nonspecific sites to adsorb proteins that will
bind to any general DNA sequence. To maximize effectiveness,
the competitor DNA must be added to the reaction along with the
extract prior to the labeled DNA target. Besides poly(dI•dC) or
other nonspecific competitor DNA, a specific unlabeled competitor
sequence can be added to the binding reaction. A 200-fold molar
excess of unlabeled target is usually sufficient to out-compete any
specific interactions. Thus, any detectable specific shift should be
eliminated by the presence of excess unlabeled specific
competitor (Figure 12). The addition of a mutant or unrelated
sequence containing a low-affinity binding site, like poly(dI•dC),
will not compete with the labeled target and the shifted band will
be preserved.
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