in vitro glycosylation

[Kyle Legate]

On 31 Oct 2001, Wolfgang Schechinger wrote:

> Any ideas how to performe glycosylation of proteins in vitro?
>
Are you trying to establish if your protein can be glycosylated?
We use glycosylation as an assay for ER membrane import. First, you need a
protein with a signal sequence and a glycosylation site (Asn-X-Ser/Thr).
Translate your protein in retic lysate (the assay will not work in a wheat
germ system) in the presence of ER microsomes (there's a Methods in
Enzymology paper by Gunter Blobel and Peter Walter that has a good method
to prepare canine pancreatic microsomes that work well with this assay).
Glycosylation can easily be visualized by SDS-PAGE, and confirmed by
treatment with endoglycosidase H to remove the sugar.
	I hope this is of some help.

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legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

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