Electrophoretic binding site is obtained, it does not

Electrophoretic
Mobility Shift Assays (EMSA); Electrophoretic Mobility
Shift Assays (also known as “band shift assays” and “mobility shift
electrophoresis”) has a standard protocol for investigating a wide range of nucleic
acid–protein interactions from single protein-binding events to assembly of
large complexes such as the spliceosome (Malloy 2000; Rio 2014). EMSA technique has been originally
introduced by Fried 1989
and nowadays many variants have been described in the literatures.  EMSA is a simple, quick, and very sensitive laboratory
technique for testing nucleic acid/protein specific interaction qualitatively,
although, under appropriate condition are used for quantitative purpose.
However, EMSA is not without limitations and more important limitations and
problems were encountered (Hellman
and Fried 2007). This technique is based on the observation that the
segments of binding nucleic acid to protein cause a decrease in the segment’s
electrophoretic mobility compared with the free nucleic acid in agarose gel under
native condition or non-denaturing polyacrylamide gel (Vinckevicius and Chakravarti, 2012; Rio 2014).
In this technique crude protein mixture or purified proteins are mixed with the
nucleic acid sequence in a suitable buffer and specific binding is allowed to
occur, stable complexes of nucleic acid and protein (the probe may be bound in
nonspecific manner by other proteins) were separated by nondenaturing gel
electrophoresis; not only for study of nucleic acid sequence requirements of binding but also
diverse aspect of nucleic acid-protein interaction including but not limited to, kinetics of binding (such as
affinity constants), identification and characterization of binding proteins,
and cofactor requirements. A wide variety of nucleic acid and protein lengths (lengths from short
oligonucleotides/amino acid to several thousand) and distinct nucleic acid structures
(single-stranded, duplex, triplex and quadruplex nucleic acids as well as small
circular DNAs) are compatible with EMSA technique (Hellman
and Fried 2007). However, with this technique, the range of DNA
sequencing binding site is obtained, it does not have enough precision in
determining the precise site and the cooperative interaction elements (for more
detail see Hellman and Fried 2007).

Deoxyribonuclease
I (DNaseI) Footprinting; A valuable technique not only for
identifying but also for characterizing DNA-protein interactions is DNaseI
Footprint method (Carey et
al. 2013). This method is also used for sequence-selective recognition
of DNA-binding ligands (Hampshire
et al. 2007). The concept is that the sequence-selective binding protein
protects the phosphodiester backbone of DNA in and around its binding site from
DNase I-catalyzed hydrolysis thus generates a “footprint” in the
cleavage ladder. DNase I which non-specifically cuts a single strand of a
double-stranded DNA helix that is not protected by e.g. binding protein,
is a convenient endonuclease for detecting and locating the position of
sequence selective binding proteins (Bailly et al., 2015). The binding sites visualized by the
autoradiography of DNA segments produced by electrophoresis on denaturing DNA sequencing gels (Brenozwitz et al. 2001; Vinckevicius
and Chakravarti, 2012). steric hindrance arising from DNA- bounded protein in the
site and its adjacent do not allow DNaseI to bind directly to the binding site
and 8-10 base pairs around it (Carey et al. 2013).

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Despite
the great value of the above-mentioned techniques, the importance of the performance
of binding proteins to these techniques is not recognizable. One solution, for
this purpose, is the use of reporter assays. Examples of it are the genes of
Chloramphenicol acetyltransferase, green fluorescent protein and assay-based
luciferase. In these techniques, the region of transcription factors in the upstream
region of a cloned reporter assay and its transcription activity is measured.
Mutations in transcription factors or their binding site make the analysis of
the sequences or important elements possible in interaction. A different
technique based on reporter assay is the formation of mono-hybrid and bi-hybrid
systems in bacteria that causes the revealing of transcription in genes because
of the interaction of many binding proteins to specific regions of DNA.