I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure.
________________________________________ De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, "Careina Edgooms" <careinaedgo...@yahoo.com<mailto:careinaedgo...@yahoo.com>> wrote: 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
