In some protocols it is advised to incubate the protein and the DNA in the binding buffer on ice, other protocols advise to perform the binding reaction at room temperature. How to manage this parameter during the binding step of an EMSA experiment?
Thanks.
I think it might depend on the protein. One way to obtain good control of your assays is to perform interaction characterization at different temperatures between the DNA and the protein to obtain information on the binding kinetics. A label-free biosensor would be the preferred choice. (E.g. Attana, Biacore or IBIS)
I agree with Teodor, I think it will be specific to each different protein and DNA interaction you are looking at
The stability of a complex between two macromolecules (protein-protein, protein-DNA), quantitatively expressed by the equilibrium association constant Ka, depends on the temperature.
Usually the enthalpy of complex formation (binding) depends linearly with temperature within an interval around room temperature, with a negative slope equal to the binding heat capacity dominated by the desolvation of hydrophobic interfaces. Therefore, there is a temperature T_h where the binding enthalpy is zero, and below and above such temperature the binding enthalpy is positive and negative, respectively. According to the van't Hoff equation the temperature for the maximal value in the equilibrium constant Ka corresponds to such temperature T_h. The temperature profile of Ka is a symmetric bell-shaped curve, within a certain interval. Of course, protein unfolding will break such symmetry and Ka will decrease at a greater extent when approaching the unfolding temperature Tm.
T_h can be determined by isothermal titration calorimetry. If T_h is unknown, a good option is to work at room temperature or 4°C (in general, T_h is usually close to room temperature, and, in general, 4°C assures folding stability).
It's been a long time since I've done this, but my recollection is that the ideal temperature strongly depends on: a) the salt concentration; and b) whether your protein binds preferentially to single stranded or double stranded DNA. Adrian's explanation is still valid, but the single-to-double stranded transition in DNA is also temperature-dependent and will alter the shape of your van't Hoff plot.
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