I am planning to do titration assay with purified His-tagged protein with biotinylated protein (purified). I need more details about getting an "apparent Kd" for interacting proteins.
The concentration of one protein (A) is held constant while the concentration of the other (B) is varied. By whatever method you choose, you measure the concentration of the protein-protein complex at each concentration of the varied protein. Then you plot the concentration of complex versus the concentration of varied protein and fit the data to a binding isotherm, such as %[AB] = 100[B]/(Kd + [B]). This only applies if the Kd is substantially higher than the concentration of the unvaried protein. If that is not true, then use the Morrison equation for tight binding situations..
You could use a pull-down of A with streptavidin beads, measuring the amount of B associated with the beads by SDS-PAGE, as one possible example.
Hi Anamika, strictly speaking, dissociation constant "Kd", only applies when you are competing one ligand binding to a compound with an identical ligand that is easily measured by isotope labeling, non-interfering additions, etc.
I think "Apparent Kd" is a bit of a misnomer, since it denotes the 1/2 concentration of a saturation binding curve regardless of what you are using to measure binding of 2 entities.
ELISA assays usually measure IC50 (50% of the inhibitor conc needed to compete). It is semantics whether IC50 is equal to Kd, but measuring an IC50 from pull-down assay is going to be difficult, unless you have a clear saturable binding curve.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence