Good points have been brought up; here's one more to consider from my experience. If you are going to run SEC prior to crystallization, I would highly recommend running a native gel of the peak you collect. Especially if you don't know the stoichiometry or if the stoichiometry is complex. I crystallized a multimeric complex where a large oligomer bound to four smaller proteins with high affinity (sub nM), and even after making the complex in stoichiometric excess, somehow one of the ligands would fall off during SEC (probably the 3-bound and 4-bound mers were in equilibrium on column) and only after I discovered this by running a native gel did I start getting single crystals instead of multi-lattice xtals. Basically the band for the full complex was fuzzy (I.e. A range of stoichiometries) and after titrating in more ligand after SEC did the band look nice. Also, you can run a native gel of your crystals (assuming you obtain some!) by harvesting a number of them in a buffer in which they'll dissolve and then use a sensitive staining method, like silver staining.
Good luck! Geoff On 12/7/12 4:00 PM, "CCP4BB automatic digest system" <lists...@jiscmail.ac.uk> wrote: >
