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
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Iain Wilson Institut für Chemie
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