I want to calculate the dynamic binding capacity and dissociation constants of a gel matrix using a standard protein, anyone has the protocol to calculate the above parameter.
Also, at how much percentage breakthrough the dissociation constant has to be determined for the accuracy and why?
Note: Weak binding is expected for the above case, may be micro molar.
I'm not sure what kind of gel matrix you are concerned with, but I'll suggest mixing a specified amount of the material with a known amount of protein, then pelleting the material with a centrifuge or, if necessary, an ultracentrifuge. Compare the protein concentrations in the supernatant with and without the gel matrix to see how much was bound. This is an equilibrium method, so you could use it to measure the dissociation constant.
Can you try Surface Plasmon Resonance method to determine the dissociation constant.
Surface Plasmon Resonance (SPR), Elipsometry, QCM and some other methods. Publication on SPR in attachement.
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