Enzymatic deglycosylation of glycoprotein

Warren Gallin wgallin at gpu.srv.ualberta.ca
Sun Jun 19 14:01:26 EST 1994


In Article <2tvhon$gc9 at news.iastate.edu>, robby at iastate.edu (Cyril S
Roberts) wrote:
>I have been trying to deglycosylate Chlamydomonas reinhardtii periplasmic
>carbonic anhydrase, using Glycopeptidase F (E.C. 32.2.18 or 3.5.1.52) from
>SIGMA.  So far I haven't been able to get it work.  This glycoprotein is a
>heterotetramer, with a number of N-asparagine linked oligosaccharides on two
>of the subunits.
>
>Essentially, I treat the protein with DTT, then boil with SDS to denature
>it.  I then incubate in 250 mM sodium phosphate buffer, along with
>glycopeptidase, overnite at 37 degree centigrade.  Subsequent SDS-PAGE does
>not reveal any effect of the enzyme.  I'd be happy to supply any further
>details as may be required for constructive criticism. 


The only thing that I can see is that you don't mention removing the SDS. 
That could be denaturing the EndoF.  Have you gat a positive control to work
under the same conditions?  One way to deal with this is to add NP-40 or
Triton X-100 to 0.5% to complex up the excess SDS.  When I was dfoing these,
about 10 years ago, the suggextion I got from John Elder was to denature in
6M guanidinum hydrochloride.
Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca




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