protein deglycosylation

C. Constantinidou c.constantinidou at bham.ac.uk
Fri Nov 17 07:21:08 EST 1995


   I have a problem deglycosylating a bacterial protein.  The protein is
glycosylated according to results obtained from the Oxford GlycoSystems
Glycotrack Carbohydrate Detection kit (periodate oxidation/biotin
hydrazide method). I have tried both chemical and enzymatic methods to
deglycosylate  partially purified protein. The chemical method used was
the GlycoFree degylcosylation kit utilizing TFMS, 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.


    Unfortunately I had only two attempts using the chemical kit before
the VERY expensive reagents run out.  So I can't speculate on what went
wrong.  With the enzymatic method I think the problem may be failure to
sufficiently denature the protein before treating it with the
glycosidases.  Currently I am denaturing by boiling the sample with SDS
and mercaptoethanol. 

One more thing.  The controls work in the presence of my protein
preparation so the presence of inhibitors in the preparation is not
likely.

 Any suggestions on what to try next would be appreciated.

Thank you,

Chrystala



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