imidazole

[Michael Witty]

Dear Artem, Patrick, Isabelle and Richard,
thanks for your replies. I have
tried IMAC with imidazole filtered and non-filtered but my instinct (being
a molecular biologist) is to autoclave anything I can in a compulsive and
probably self destructive way. Bottom line: even filtering was not
necessary. I still feel dirty. Mike.

On Sun, 29 Apr 2001, Dr. Artem Evdokimov wrote:

> Generally it might not be the best thing to do, especially if you have
> traces of transition metals in your solution. You can expect some
> hydrolysis if you decide to autoclave concentrated imidazole solutions -
> whether this would affect the behavior of your IMAC - I can't predict
> w/o trying. The one time I autoclaved 300 mM imidazole at pH 8 it turned
> slightly yellow.
> My guess would be that even if some imidazole decomposes/polymerizes,
> there will still be enough of the intact compound left to work with. I
> am not sure what these decomposition products might do to your protein,
> though.
>
> If you were to autoclave things, I'd say that pH 5 would be safer than
> pH 8 but that's just a guess. Since most IMAC procedures use pH 8, you'd
> still have to adjust your pH in the end so autoclaving at pH 5 makes no
> sense.
>
> What's wrong with sterile filtration, anyway ?
>
> Artem
>
> Michael Witty wrote:
> >
> > Dear All,
> > what are our opinions on autoclaving imidazole in buffers for
> > Ni-NTA? I think we shouldn't because this would cause imidazole
> > decomoposition. Is there any experience out there? Mike.
>
> --
> |Dr. Artem Evdokimov Protein Engineering |
> | NCI-Frederick Tel. (301)846-5401 |
> | FAX (301)846-7148 |
> | eudo...@mail.ncifcrf.gov |
> | http://www.ncifcrf.gov/plague |
>

[Dr. Artem Evdokimov]

Hi,

Glad to help.

Actually filtering is necessary if you are working with any kind of FPLC
or HPLC system, or using expensive fine-grain columns (such as the
'monoX' series, hydroxyapathite, 5u C-X and CN-X series, superdex etc.).
Our AKTA's really appreciate filtered solutions - in fact everything we
put on them is filtered, including cell lysates. It helps a lot - you
won't believe what kind of junk one finds in commercial salts and
chemicals, even in the 'super-duper clean' ones (most common are fibers
from some kind of sacking, it would appear). Autoclaving does not
eliminate all particulate matter, so if you are working with any kind of
precision machinery, expensive columns etc. then 0.22 u filter is your
best friend.

We are mostly dealing with proteins being prepared for crystallographic
studies so as you can imagine, the amounts of liquids that we put
through the systems are quite large, but even for small samples
sometimes just one piece of fiber is enough to crack your column or
increase the backpressure or get stuck in your pumps and
cross-contaminate your sample.

Cheers,

Artem

Michael Witty wrote:
>
> Dear Artem, Patrick, Isabelle and Richard,
> thanks for your replies. I have
> tried IMAC with imidazole filtered and non-filtered but my instinct (being
> a molecular biologist) is to autoclave anything I can in a compulsive and
> probably self destructive way. Bottom line: even filtering was not
> necessary. I still feel dirty. Mike.

--

[Dr. Artem Evdokimov]

> And I'd do it to protect the - not inexpensive - Ni-NTA resin.
Shamefully expensive, knowing how cheap mass manufacture is !

>
> > We are mostly dealing with proteins being prepared for crystallographic
> > studies so as you can imagine, the amounts of liquids that we put
> > through the systems are quite large, but even for small samples
> > sometimes just one piece of fiber is enough to crack your column or
> > increase the backpressure or get stuck in your pumps and
> > cross-contaminate your sample.
>

> And one piece of dust could give you a crystal you never see again . . .

There's plenty of opportunities to collect dust for xtallization
purposes after chromatography - just look inside some of the microvial
types :) (yes they're sterile but DUSTY!)

> BTW Michael, if you are doing this for crystallization and not getting
> anywhere, you might try the same protein sans His-tag.

Or move the tag onto the different end of the protein. There are too
many answers to the question 'why my protein does not crystallize' :)
(and a sad lack of answers to the question 'how do I crystallize a given
protein'.

Artem

[Michael Witty]

> > and a sad lack of answers to the question 'how do I crystallize a given
> > protein'.
>

> A sad lack of *helpful* answers :-))

But the happy ending is that I eventually got 0.52mm long crystals. And
you are right, the plasticware I used was full of dust! Mike.

[Dr. Artem Evdokimov]

> But the happy ending is that I eventually got 0.52mm long crystals. And
> you are right, the plasticware I used was full of dust! Mike.

Congrats !

What really bites is that only about 6-15% of all proteins that are
cloned more or less indiscriminately (as in 'structural genomics') ever
crystallize. We have a sample of about 40 proteins but don't take my
word for it - recent results of the structural genomics consortia show
the same trend.

We have a pretty good methodology to bring the proteins that show
crystallinity to the final stage (i.e. well behaved single crystals) but
the problem is still getting this first hit.