Huseyin Kucuktas <kucukhu at mail.auburn.edu> wrote:
>>> Hello,
>> I am planning to use glycoprotein detection/differentation kits
> to analyze a glycoprotein from a virus.
>> How efficent they are working?
> How much glycoprotein I should use?
> Any other comments...
>> Any kind of reply would be appreciated!
Hope my answer isn't too late - but I don't read bionet.immunol
regularly.
Depends on the sort of glycosylation you're looking for - is it
N-linked, O-linked mucin type or O-linked GlcNAc or don't you know?
Very simple detection of oligomannose, most hybrid, and biantennary
complex N-linked oligosaccharides is possible with Concanavalin A
after blotting - just use normal SDS-PAGE - blot, block, wash as
normal and then use 50 micrograms per ml Concanavalin A (ConA) with
2.2 mg CaCl2, 3.2 mg MnCl2 (in 20 ml), rock 2 hrs - wash twice and
add 1U/ml horseradish peroxidase (100U is approx 1 mg) with 2.2 mg
CaCl2 and 1 mM MgCl2 (20 ml). Wash twice and develop colour with
30 mg 4-chloro-1-naphthol in 10 ml MeOH, 40 ml PBS, 30 microlitre
hydrogen peroxide. Stop reaction with 5% acetic acid in water.
(Note horseradish peroxidase has stuctures which bind ConA - one
relies on the multivalency of ConA to effect detection.)
However - some N-linked structures may not and O-linked structures
will not bind to ConA. However this ConA method would probably be
significantly cheaper than an OGS or Boehringer kit.
Overall, your question could also be asked on the bionet.glycosci
group.
Iain
---------------------------------------------------------------
Iain Wilson Institut für Chemie
Tel: 43-1-47654-6065 Universität für Bodenkultur
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E-mail: wilson at edv1.boku.ac.at A-1180, WIEN, Austria
http://www-bprc.mps.ohio-state.edu/cgi-bin/hpp/iain_wilson.html
