I was always told that gel filtration resins have a mild ion-exchange character, hence the recommendation to use at least 100mM NaCl in size exclusion buffers. Assuming that it is true, one would expect a protein to stick to the resin in low salt buffers. That is the opposite of what you see (your protein elutes earlier in low salt buffer). That makes me think that you are actually experiencing some sort of oligomerization and/or shape transition. Do you have access to MALS? It would give you a definitive answer.
On Tue, Mar 22, 2011 at 8:23 PM, Jacob Keller < j-kell...@fsm.northwestern.edu> wrote: > Dear Crystallographers, > > I have run my protein-peptide complex several times on a GE S200 > 10/300 in buffer A (below). Today, to make a crystallization stock, I > ran the sample in buffer B, and the peak shifted from a consistent > 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that > SEC results change as a result of buffer conditions. Could this > drastic a shift be due simply to buffer conditions, or could there > actually be some buffer/ion-dependent dimerization going on? Anyone > have a similar experience? > > A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) > B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) > > Jacob Keller > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ******************************************* > -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
