Cell Culture - How to isolate membranes ?

[f.r.byrne]

Hello World,
Hope you can help us with a problem here.

We are trying to isolate a receptor for the Botulinum neurotoxin
and we have several cell lines that we and others can demonstrate
 binding to(specifically the human and murine neuroblastomas
 GO-G-GCM, SK-N-SH and best ofall NIE-115) and control lines that it
 does not (e.g. the Vero line).

We are trying to isolate whole membranes from these adherent
 cultured lines with a view to getting our hands on the receptor
 for the neurotoxin (not the intracellular substrate synaptotagmin).
 Have tried various percoll and sucrose gradients based on the methods
 of Dunkeld in Brain Research (1994) and we get nice banding patterns
 with percoll but no fractions show any marker activity for membrane
 enzymes - we looked for 5' Nucleotidase in a liquid
scintillation assay as detailed in 'centrifugation - a practical aproach'
by D Rickwood.

Nothing much - assay for cytochrome reductase in every fraction
 was also inconclusive although commercial enzyme shows
 assay is sound.
The membrane isolation procedure involves incubation with
iodonitroneotetrazolium violet and spinning etc.etc.
As I say, nice bands in the percoll (2 ml of 23%, 15%, 10 % and 3%)
but it seems to be all crap.

Does anybody know a good way to successfully isolate and assay for
whole membranes from adherent mammalian cell culture neuroblastoma
cell lines like these ????

Help gratefully received.
Tel. ; 01224-877071 ext. 212 or 
e mail f.r.byrne at abdn.ac.uk
Thanks in advance,

Fergus Byrne
Aberdeen, Scotland