Cell Culture - How to isolate membranes ?
gen126 at abdn.ac.uk
Thu Jul 6 08:46:29 EST 1995
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,
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