Hi Jerry -
By AUC, do you mean sedimentation velocity (SV)? Both gel filtration and SV are not terribly great ways to determine precise molecular mass, especially if the macromolecule of interest is anisotropic in shape. In your SV values, do you see a large f/fo, or a broad distribution? Can you run a sedimentation equilibrium experiment? If you run HYDROPRO on your prospective oligomer structure, do you arrive at theoretical S and Rs values that jive with your solution data? A nice crosscheck you could do with the data in hand (if your measurements from both approaches were performed in the same buffer at the same temperature) is calculate the mass using the Siegel and Monty equation (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346-362), where the mass of the particle is calculated from Rs (from gel filtration) and the S(t,b) value from sedimentation velocity. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine <BLOCKED::mailto:kgu...@stwing.upenn.edu> kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state. Could anyone give some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully
