Hi Acoot, since they behave differently on IEX, they are different - you would introduce heterogeneity into your crystallization setup, which usually is not a good idea for crystallization in general. Are you using Benzonucleases by any chance in your preparation and if not, that may explain the different populations of your protein. Your protein may bind some short DNA chunks carried over during your lysis process leading to differently charged species on the IEX column. On an SDS gel these complexes are separated and your protein runs at an identical molecular weight. An alternative explanation would be multiple folded states that may be at equilibrium and under certain conditions e.g. pH, salt concentration may be pushed in one predominant species. Also is several 2 or 10 peaks ? If you have e.g. an N-terminal His6-tag you might end up with truncation products and say your protein is rather large e.g. 80 kDa you might not be able to see the molecular weight difference either by SEC or SDS-PAGE if you are running a low percentage gel. A protein of 72 kDa might look like 80 kDa but you most likely will have charge differences distinguishable in IEX. If you have a His6-tag, run a Western on it to identify single or multiple bands.
Good luck, Jürgen On Dec 14, 2013, at 7:16 AM, Acoot Brett <acootbr...@yahoo.com<mailto:acootbr...@yahoo.com>> wrote: Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
