Preparative separation of a crude O-glycan mixture

Iain Wilson wilson at edv1.boku.ac.at
Tue Apr 11 05:44:50 EST 1995


Andreas Kage <akage at fub46.zedat.fu-berlin.de> wrote:
>
> I have a crude mixture of O-glycans from a heterogenous glycoprotein 
> preparation after beta-elimination. What is a good procedure for 
> preparative separation to find a fraction that 
> interacts with my lectin-like protein. 

It depends on what property you wish to use - size, charge, type, etc.

A reasonable size fractionation method is to use Bio-Gel P4 - with a 
decent HPLC pump, fined gel, pressure overload valve and pre-column 
for the degassed/helium sparged water input (if you have decent 
amounts of material a refractive index monitor would be good for
preparative work). Some people use P4 with gravity but I believe it 
takes a very long time; others have difficulty with P4 columns - you
just have to invest the time, effort and some money in it. I 
successfully set up such a P4 system at my previous lab for N-linked 
oligosaccharide analysis (after one or two problems) and the columns 
run OK for 12-18 months. It is important to have a reliable pump and 
to change the guard column (ion exchange resins) every so often.

For charge fractionation - some DEAE or MonoQ columns can be used.

There are other techniques such as HPLC on amine bonded silica or 
reverse phase - I have no experience of these.

In the Glycobiology volume in the IRL Practical Approach series
there is an article from Piller and Piller on O-linked 
oligosaccharide fractionation and characterisation.
 
I am sure there are people out there with direct experience with 
O-links - so share your knowledge with us.

Iain

---------------------------------------------------------------
Iain Wilson                        Institut für Chemie           
Tel: 43-1-47654-6065               Universität für Bodenkultur   
Fax: 43-1-310-5176                 Gregor-Mendel-Straße 33
E-mail: wilson at edv1.boku.ac.at     A-1180, WIEN, Austria

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