In Article <2tvhon$gc9 at news.iastate.edu>, robby at iastate.edu (Cyril S
>I have been trying to deglycosylate Chlamydomonas reinhardtii periplasmic
>carbonic anhydrase, using Glycopeptidase F (E.C. 32.2.18 or 188.8.131.52) 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.
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca