At 07:23 PM 3/22/2011, Jacob Keller 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.)
So, it elutes earlier in essentially zero salt. I would bet that the
protein is acidic and what you see is a buffer effect. Superdex (and most
other gel filtration matrices) carries residual negative charge. So in
"zero" salt there will be repulsion between protein and beads, resulting in
the protein entering pore less frequently. Hence the earlier elution. I've
seen this effect for a couple of monomeric acidic proteins. Chances are,
switching to a salt higher than 50 mM will also retard the elution a bit.
Typical recommended salt in gel filtration is in 100-200 mM range precisely
to suppress ionic interactions.
- Dima
