Dear Steve,
I don't have any experience of the Promega kit you mention but I can help
on the N-glycosylation stuff.
All eukaryotes seem to share the early steps of N-glycosylation. They add a
preformed glycan to the sequence Asn-Xaa-Ser/Thr. This is done
co-translationally in the ER. So if your protein has an appropriate
sequence the canine microsome preparation should N-glycosylate it. There
are differences between the mature structures on animal, yeast and plant
glycoproteins but these are due to processing events and additions
catalysed primarily by Golgi enzymes.
I think your experiment should work as you describe it. To check that any
shift in mol. wt. id due to N-glycosylation you could check for
susceptibility to Endo H. But that's another story.
Dave Ashford
*************************************************************************
Dr David A. Ashford
Glycobiology: Research & Analytical
Department of Biology
University of York Tel: (44)(0)1904 434391
P.O. Box 373 Fax: (44)(0)1904 434312
York YO1 5YW Email: da5 at york.ac.uk
*************************************************************************
On 15 Apr 1997 14:26:05 GMT Steve Dunn wrote:
> From: Steve Dunn <dunnsm at bbsrc.ac.uk>
> Date: 15 Apr 1997 14:26:05 GMT
> Subject: in vitro glycosylation
> To: "bionet.glycosci mail newsgroup" <bionet-news at dl.ac.uk>
>> Hi!
> I'm wondering whether anyone's familiar with Promega's T7 TNT quick
> kit for in vitro transcription/translation. I wish to use it in
> conjunction with their canine microsomes to investigate possible
> glycosylation/processing of my gene which encodes a membrane protein.
> The catch! My protein is from a plant not mammalian. So, if I see any
> glycosylation, is it likely to be relevant and a good indication of
> what might be occurring in the plant ie. in vivo (I accept the nature of
> the glycosyl moiety is likely to be different, it's just the occurrence
> I'm conserved with). I guess I'm really asking whether glycosylation
> recognition sites are conserved between eucaryotes. Can anybody shed
> some light on this ?
> Many thanks for any enlightening comments,
> Steve
>>dunnsm at bbsrc.ac.uk
