In article <Pine.3.89.9501301624.A28223-0100000 at ccshst01.cs.uoguelph.ca>,
aiyo at uoguelph.ca (Abiye Iyo) wrote:
> Hi,
> I recently used the GST fusion system to purify my protein (a
> cellulase). Everything went fine up to the stage of gel filteration and
> I had a nice single band . Now the misery part is that my protein which
> is about 57kD degrades to two low molecular weight products which are
> still active on zymograms. Does this mean my prep is contaminated with
> proteases or I am experiencing some form of self digestion judging from
> the fact that my protein was pure initially? And how can I prevent
> this? I need help before my patience runs out.
>> Abiye
You should also check for bacterial contamination. Gel filtration columns
(especially Sephadex) are notoriously good at growing bugs - I assume you
are using a bacteriostatic or bactericidal agent to store the column when not
being used. Phosphate and acetate buffers should of course be sterile
prior to use also.
Nick Fisher
Biochemistry Dept
Cambridge University
