purification Ig like domains

R. Woodward rw200 at cus.cam.ac.uk
Mon Dec 15 12:27:40 EST 1997

Dear All,

I am despirately trying to purify the extracellular domains of tyrosine
kinase receptors (PDGF like) which are secreted into my culture media.  At prese
nt I am using cationic exchange resin (Resource S from Pharmacia) my major probl
em is that my product does not elute as a sharp peak, rather it appears
to elute over a broad salt concentration and is not confined to a single peak.
I have tried loading and eluting slowly, decreasing the salt gradient,
detergents and glycerol in unsuccessful attempts to cure this problem.
Has anyone got ideas as to why I should see this characteristic?
Is it due to my protein or the exchanger?  Has anyone had similar problems?

Two other points has anyone had experience of purifying Ig Fab arms other
than by using affinity chromatography? Do you need to add any reducing agents
to prevent breakdown of the 2ndry structure.

Also is there any milage in using other salts e.g. KCl rather than NaCl
when eluting from an ion exchange column?

Thenks in advance    Robert
Email rw200 at cus.cam.ac.uk

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