Glycoprotein analysis kits

Iain Wilson wilson at edv1.boku.ac.at
Mon Mar 27 11:46:20 EST 1995


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   
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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