mw132 at mole.bio.cam.ac.uk
Mon Apr 30 04:45:56 EST 2001
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,
> 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
> What's wrong with sterile filtration, anyway ?
> 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 |
> | eudokima at mail.ncifcrf.gov |
> | http://www.ncifcrf.gov/plague |
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