Hi! I am doing some EMSA and got some questions on the basics (not much experience in biochem), could anyone give me some help? Many thanks!
So why do we usually use low DNA concentration in this kind of assay (usually below Kd as I found)? What will happen if we titrate a DNA at, for example, 1 micromolar with protein, and the DNA can be directly stained by ethidium bromide or stains-all? Is it possible we get Kd from such an EMSA gel at such high DNA concentration?
First, EMSA is not adequate to determine the Kd of the interaction, since the concentration of free protein around the DNA is going to be variable and drop as soon as the protein and the DNA are separated in the course of migration. Secondly, since it is the DNA that is monitored, its concentration is irrelevant; what matters is that the concentration of protein should be above the Kd. Using a DNA concentration above the Kd would be important if you were to monitor the migration of the protein.
Thanks! What I am reading is this paper:"Using Electrophoretic Mobility shift Assays to Measure EquilibriumDissociation Constants: GAL4-p53 Binding DNA as a Model System" http://onlinelibrary.wiley.com/doi/10.1002/bmb.20649/abstract
As it says, " To measure the KD for a protein/DNA interaction, a series of binding reactions are performed in which the concentration of the DNA is below the KD for the interaction, and the protein is titrated from below to above the KD. This requires an initial estimate of the KD, which can be determined from a preliminary experiment in which the DNA concentration is set low and the protein is titrated over a broad range." I don't fully understand this part. If normal DNA concentration is acceptable, why do we bother to use radiolabelling or fluorophore for EMSA?
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