Hello,
I'm working on nanobodies that incorporate propargyl-lysin (PrK) in its sequence. I observed that most of my protein was in dimer form prior to loading it to the antibody column, and that in this form, the nanobody was unable to bind to it and was lost. The dimers aren't formed by disulfide bonds, and I don't know what kind of interactions the PrK might have that would cause them. I would appreciate any assistance from anyone who has experienced such dimers with PrK.
Have you tried to positively identify Prk as the culprit? You could do a click chemistry derivatization with some organic azide (e.g. N3-CH2-CH2-OH or -NH2) before you go onto the column, and see what happens.
Otherwise, try changing pH, adding NaCl, MgCl2, urea, etc.
If the affinity column works with the unmodified nanobody, it might be a paratope problem, though, i.e., The propargyl residue is masking the binding site.
In addition to the above responses, you may also test glycine alone and non-denaturing detergents such as triton x-100 at low concentrations to disrupt the oligomerization affinity. But you should be aware that, some of the abovementioned suggestions (including triton) will hamper affinity-induced protein purification...Therefore upon breaking the aggregate, techniques such as SEC, UF, Dialysis, gravity flow desalting/buffer exchange may get rid of these agents prior to successful affinity chromatography.
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