In article <32AEF369.6AF9 at ensam.inra.fr>, urbach at ENSAM.INRA.FR (urbach) wrote:
> Hello all,
>> I try to determine if a protein is glycosylated using SF9 expression
> system.
> I test tunycamicine effect on N-glycosylation.
> To test if "my" protein is o-glysosylated, I would like to use an
> specific inhibitor, like neuraminidase. But is it a good inhibitor?,
> Witch concentration have i to use?
> Excuse for my question, and my english but i am not a specialist.
> Thank you for answer directly to my e.mail adress
>> Serge URBACH
>urbach at ensam.inra.fr> Laboratoire de Biochimie et physiologie moleculaire des plantes
> ENSAM/INRA/CNRS/UNII
> Montpellier
Neuraminidase is not an inhibitor. It is an enzyme specific for cleaving
sialic acid from galactose. In a biochemical assay, it will not tell you
whether your protein is o-glycosylated. I am not aware of any 'good'
o-glycosylation inhibitors available now. Alternatively, you can labelled
your protein with tritium (3H) in the presence of inhibitors of
n-lycosylation and examine whether your protein is still radiolabelled. If
it is, it must be on the o-glycans. Or you can 3H label your protein under
normal condition, deglycosylate the protein with PNGase and detect
residual radio signal and band shift.
Wei Wu
