In article <01bcbef5$9e16f1a0$30019386 at schmidtdw.rz.ruhr-uni-bochum.de>,
Thorsten Schmidt <Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:
>>I now want to test whether it is glycosylated or not!
>How can I do this? What do I need?
If it's N-glycosylated, treatment with endoglycosidase F will cleave the
glycoproteins and you'll see the MW of the protein drop. If it's
O-glycosylated ... someone else will hae to tell you what to use because I
can't remember.
EndoF is available from various places; I most recently got it from New
England Biolabs, which calls it "peptide: N-glycosidase F (PNGase F)".
>Are there enzymes/kits availible for deglycosylation?
NEB sells it as a kit with instructions. It's very simple.
>Are there different types of protein glycolysation and can I examine
>them all just with one test?
There are many kinds of N-glycosylation, but as far as I know EndoF will
strip off all of them. You can refine the analysis with other enzymes, if
you want.
As for O-glycosylation, that's less likely to be causing the MW shift you
see (in my humble and no doubt ill-informed opinion), so you'll want to
start with Endo F.
>Can I conclude protein glycosylation just from special aminoacid sequences?
You can make guesses. You need to see a signal sequence (because the
protein has to be transferred into the endoplasmic reticulum for the
glycosylation machinery to reach it) plus either NXT or NXS. The presence
of these does not guarantee that they'll be used, but if you see these in
the presence of a MW shift the odds are pretty good that they are used, I
think.
Hope this helps.
Ian
--
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
