Dear Carina, An additional problem is that due to the dilution, but also due to the separation of monomers and dimers you get a reequilibration which is dependent on Kon/Koff of the interaction. Unless these are very slow, you cannot use size exclusion to determine the monomer/dimer ratio. Although not perfect, I would try dynamic light scattering. For calculating the Kd, I would just use the standard textbook formula, with A being identical to B.
Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina Edgooms Gesendet: Freitag, 14. Februar 2014 09:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] KD of dimerization, off topic Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
