James Fethiere (James.Fethiere at BRI.NRC.CA) wrote:
: Hello all, I am using different endo and exoglycosidases from
: Boeringher to deglycosylate glycoproteins. I usually put an excess
: of enzyme but I would like to know if the is specific amounts of these
: enzymes to use. What i need is a number referring to mg enz/mg of glycoprotein.
: Or in some cases U enz./mg protein, although this unit thing is sometime
: confusing.
: Thank you
I think that this sort of thing really has to be determined empirically,
as the mass of glycan:peptide varies. My approach would be to start with
as little as possible (10 times less than manufacturers recommend) and
titrate up from there (easy to do if you have a visible gel shift assay).
Also, do you denature the target glycoprotein first? I knbow that BM
recommend the use od nonidet NP-40 and trace SDS to 'open' up the target
protein prior to use of PNGaseF (Glycopeptidase-F). If the study is
purely structural, with no need for activity of target protei afterwards,
then why not try reduction and acetylation of the target protein in
poorder to allow easy access of the glycopeptidase?
--
__________________________________________________
Steven Pirie-Shepherd
srps at galactose.mc.duke.edu
"Insert your own pithy phrase just about here!"
