Deglycosylation protocols

[Ian McFarlane]

In article <firths-2304981119090001 at 128.250.234.75>,
firths at cryptic.rch.unimelb.edu.au (Steven Firth) wrote:

> Hi, I'm after protocols for the deglycosylation of whole cell proteins
> from a mammalian cell pellet. I'm currently using PNGase F from NEB but on
> whole cell proteins I am getting substantial degradation of protein.
> Any suggestions would be welcome.
> 
> Stephen Firth
> Trace Element Laboratory
> The Murdoch Institute for Research into Birth Defects
> The Royal Children's Hospital
> Melbourne, Australia.

With a whole cell pellet you will have use some protease inhibitors in the
mix to prevent the contents of the lysosomes degrading your glycoproteins.
Another (more complicated) way to avoid this happening is to prepare
isolated cell membranes in which the lysosomal protease content is greatly
reduced.

Ian Mc