Hi Dima, I forgot to mention that previously, I had coupled the ligand to BSA, previously immobilized to sepharose, with a similar overall ligand density (about 50µmol in 10ml of sepharose). There I could elute about 15% specific IgY in a similar exploratory experiment, So I doubt unspecific binding of IgY. However, I also observed some ion exchange properties of the BSA-derived material, so instead we decided to try direct coupling of the ligand to a (appropriately modified) sepharose. At the moment, I favor your "too high coupling density" theory. I haven't tried stronger chaotropes like NaI and NaSCN yet, so I am curious, what results I will get on monday. NEt3, pH 10.5 is an additional another option. And making a smaller column which might be better suitable for method development. In worst case, I'd revert to the BSA based column.
Concerning IgY, it's incomparably cheap compared to mice and rabbits and we usually get good results. Sheep and goats are reasonable alternatives, of course, and we also use them. However, I already have about 500mg of crude IgY waiting in the fridge for purification, so changing the animal is not an option anymore. Many thanks for your help! Wo > Sounds like any IgY bind strongly to your column. Which means > you are doing non-specific absorption chromatography rather than > affinity. Two possible reasons: 1) your conditions or 2) your ligand. > #1 is unlikely unless you keep screwing up royally, #2 is very likely > if your ligand is at very high coupling density (you hint at that with > your stated high theoretical capacity) and #2 is possible if your > ligand is unusual in that IgY bind to it strongly (is it something > strongly hydrophobic or soemthing likely to bind complex sugars?). > More exotic case is that you did not block sites left over after coupling > chemistry and you are doing covalent chromatography. > > I'd try: batch chromatography versus column AND lower coupled > ligand concentration 10 and 100-fold and see what happens. If > I can use some derivative of that ligand (e.g., deoxy form or > something), I'd try coupling that, too. > > As a general comment, forget chickens, go with rabbits or goats. > Way easier and higher affinity antibody. > > DK _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
