fhoedem at oci.utoronto.ca
Sun Nov 19 14:05:46 EST 1995
C. Constantinidou wrote:
> I have a problem deglycosylating a bacterial protein. The protein is
> glycosylated according to results obtained from the Oxford [...], and > the enzymatic method
> was the GlycoShift de-N-glycosylation kit utilizing both
> peptide-N-glycosidase F and a mixture of Endo-F/peptide-N-glycosidase F.
> Both kits were purchased from Oxford GlycoSystems. The control proteins
> supplied with the kits have always shifted molecular weight (on SDS-PAGE)
> after treatment, as expected. The protein of interest has failed to do so.
The first thing that comes to mind is O-linked glycosylation on serines
and/or threonines. Your kit will not cleave these. I do not know if
there are kits for deglycosylation of O-links, but I know Boehringer
sells a whole bunch of glycosylases.... (No commercial interest for me)
BTW, what bacteria, and how do you express your protein?
Hope this helps, Flip
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