![]() The only requirement is to use protein and ligand concentrations in the range of Kd so that you can actually see binding variation when total protein concentration varies (in your experiment it is expected a nanomolar Kd).Īnswering your questions (things may change depending on your answer above): The approximation X=total protein is made when total protein is much greater than total ligand. So in the equation if Y is bound DNA then Bmax is total DNA and X has to be free protein (total protein minus bound protein). ![]() In your case of EMSA, you easily spot and quantify bound and free forms of DNA on gel and knowing protein has a single binding site for DNA, it means that bound DNA is also bound protein. For the latter, there is no absolute need to have a large excess of one of the components as long as you can identify bound and free forms of the ligand or of the protein. ![]() I think you are doing a mix between enzyme activity determination where it is important to work in large excess of substrate (in order to observe initial velocities for Km, Vm determination) and condition for Kd determination for protein ligand interaction. ![]()
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