Biomod/2011/Slovenia/BioNanoWizards/methemsa

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EMSA



EMSA stands for Electrophoretic Mobility Shift Assay. It is a general term describing an assay where complex formation is observed with electrophoresis. A complex generally has different mobility in an electrophoresis gel than each of its components alone. This is noticed as a change or a shift in band position in an electrophoresis gel in comparison with its components. It is widely used in life sciences, most notably for analyzing DNA binding sites for transcription factors or other proteins, such as our chimeric zinc finger proteins. We used agarose gel and ethidium bromide staining to detect oligonucleotides and the complexes they form with zinc finger proteins. If such protein binds to DNA, the resulting complex is still negatively charged but of higher Mw than an oligonucleotide alone, therefore the oligonucleotide has an apparently higher Mw according to a ladder.

Samples for EMSA were prepared by adding different molar excesses of protein to 0.5 μl of 20 μM binding oligonucleotide solution and a buffer (20 mM Tris pH 7.6, 100 mM NaCl, 5 mM TCEP, 0.1 % BSA) up to a final volume of 27 μl. Protein was added 4-fold, 8-fold or 16-fold molar excess over binding oligonucleotide. One of the samples contained the same amount of oligonucleotide as the mixed samples, but no protein was added. Another contained the same amount of protein as the sample with the highest molar excess of protein, but without addition of oligonucleotide. After 1 h at room temperature 3 μl of 50 % glycerol were added and 25 μl of each sample were loaded onto a 2 % agarose gel with ethidium bromide and run for 50 minutes at 70 V in 1x TAE running buffer. DNA was visualized with UV illumination and photographed.

 

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