in vitro glycosylation
legatek at mcmail.cis.mcmaster.ca
Wed Oct 31 17:09:39 EST 2001
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
Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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