Stabilizing labile DNA-protein complexes in polyacrylamide gels.
The electrophoretic mobility-shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K(a)>10(9) M(-1) under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.
Sidorova
Nina Y
NY
Laboratory of Physical and Structural Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. sidorova@mail.nih.gov
Hung
Stevephen
S
Rau
Donald C
DC
D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D046911:Macromolecular Substances / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; C028914:triethylene glycol; D011092:Polyethylene Glycols; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D005990:Glycerol