IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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




More information about the Chlamy mailing list

Send comments to us at biosci-help [At] net.bio.net