Do proteins self-digest?
nef at mole.bio.cam.ac.uk
Wed Feb 1 12:25:00 EST 1995
In article <Pine.3.89.9501301624.A28223-0100000 at ccshst01.cs.uoguelph.ca>,
aiyo at uoguelph.ca (Abiye Iyo) wrote:
> 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.
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.
More information about the Proteins