imidazole buffer has esterase activity

[Zophonias O. Jonsson]

In article <32F975D7.6375 at probes.com>, dick at probes.com wrote:

> As an organic chemist I would not be at all surprised by imidazole
> catalyzed hydrolysis of p-nitrophenyl butyrate.  It is an example of
> general base catalysis of hydrolysis of an ester.  p-Nitrophenyl esters
> are hydrolyzed at a moderate, pH-dependent rate in any buffer and are
> VERY unstable about pH 9.  You MAY be able to use N-methylimidazole,
> whose mechanism of hydrolysis could be a bit different than imidazole. 
> Also avoid any buffers containing primary amines such as Tris.

Another possibility is to leave out the Imidazole altogether.  You can
elute proteins from Ni-NTA colums (and other Ni-chelating columns) by low
pH.  If your enzyme can take it, it may be the easiest way.  Alternatively
moderate amounts of EDTA will strip everything away from the column. 
Histidine could also be a candidate for elution but may not solve your
assay problems.  Take a look at Quiagen's IMAC handbook.  They also have a
web site where you may find some usefol info.

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
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