[Protein-analysis] Re: Enzyme immobilization.
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Wed Jul 20 11:38:19 EST 2005
Chris B. wrote:
> Can I and how I can bind protease (pepsin, trypsin) to chitosan or sephadex?
> I heard about binding enzyme to chitosan using glutaraldehyde and to
> sepharose using cyanogen bromide.
> Does anybody have good and cheap method to share with protein science
> freshman? :)
You can buy cyanogen bromide activated sepharose from Pharmacia, and
from several companies that sell it repacked under their own label. This
is much safer than messing with CNBr, which is a highly toxic, volatile
liquid. You just swell the gel and incubate it with your protein, as by
manufacturers instructions. Unfortunately, pre-activated gels (either
CNBr or other chemistries) are not cheap.
Another way to immobilise proteins is to pass them through a PVDF
blotting membrane, for example in a syringe filter. Many enzymes survive
binding by hydrophobic interactions intact. Then pass the substrate
solution through the filter. I have once used this approach for rapid
(non-steady state) kinetics. Hint: do not use nitrocellulose membranes,
although they are cheaper than PVDF. Protein binding capacity is lower
and protein leakage into the substrate higher.
> And one more thing - what is stability of such an enzyme? After how much
> time or how many use it looses activity (below "resonable" level)?
That depends on the protein, the immobilisation method and the storage
conditions. No hard and fast rules, only experiments will tell. With a
quick and cheap method like the PVDF membrane and cheap proteins like
pepsin I'd not sweat it and just make a fresh one each time. Although of
course you'd have to document the stability of your preparation, at
least for the time of the experiment.
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