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Plenty of possible reasons for this :) But then,
you're not telling us several important pieces of data - e.g. what else was in
there with the protein, how the protein was concentrated, what was the protein
theoretical pI, what was the protein size, was there *any* amount of protein
that actually bound to the column - or did it *all* go through, what column
material did you use, etc.
Without these data all we can offer is mostly
speculation.
Three possibilities likely:
1. you had unexpected co-solute together with the
protein and it prevented the binding
2. your pI was not right even at pH
8.0
3. your protein state changes with concentration
(monomeric vs aggregated or oligomerized)
Tell us more details and maybe someone will have a
more detailed explanation for you :)
Artem Evdokimov
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