imidazole and anion exchange resin
dk at no.email.thankstospam.net
Wed Jul 24 22:48:05 EST 2002
Margaret Luk-Paszyc <polshgrl at ilion.bio.sunysb.edu> wrote:
>Hello all --
>I have thoroughly enjoyed reading all of the posts and responses over the
>last few months, but I finally have a question of my own. Concerning an
>anion exchange resin, such as Amersham's source 30Q, does imidazole
>(say, 50 mM imidazole) pose a hazard to my column? If so, what
>concentration of imidazole is considered "safe". To better explain my
>situation, I am working on a 6xHis-tagged protein that apparently degrades
>the longer it sits; to combat this problem, I am trying to purify as
>quickly as I can. I've come across a similar problem as other bionetters,
>where my protein elutes from a Ni-NTA column at only 50 mM imidazole...
>and dialysis takes too long and additional bands appear. PD10 columns
>seem to eat my protein, so that is out of the question. The only other
>option I can think of is diluting my Ni-NTA pooled fractions.
>Does anyone else have a good idea, or can anyone at least tell me how bad
>imidazole is for Q resin?
Imidazole is not bad for any ion-exchanger, period. As long as your protein
binds, there is absolutely no problem. 50 mM imidazole at ~ neutral pH is
rather low ionic strenght, so most acidic proteins will bind happily while
imidazole, being either neutral or positively charged, go through.
All you really need to make sure is to omit high salt from your IMAC
elution solution. Source Q sounds like a fancy and expensive way of
achieving simple concentration/buffer exchange procedure. As capaciries
of most ion-exchanges are very high, you should be good with their "High
Trap" line of columns, perhaps even 1 ml variety (assuming you are
against using your own gravity flow column, which is by far cheaper).
If you don't mind high phosphate in the end of it all, frequently a much
more reliable way to concentrate is to load material on a small
hydroxylapatite column. Same idea as ion exchanger but you can have
high salt in loading buffer.
In either case you are going to have some loss on a column which maybe
anywhere from very small to very high, depending on protein.
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