PIPLC use

hcavaco at fm.ul.pt hcavaco at fm.ul.pt
Fri Mar 31 07:12:03 EST 1995


> I seem to have a problem with PIPLC use in culture. 
> According to a paper on the purification and properties of the PIPLC
from 'Bacillus 
> Thuringiensis', KCL and NaCL at >[10mM] are inhibitory for
PIPLC.(Biochimica&Biophysica Acta,
> 619,1980,48-57)
> I want to use it on some brain endothelial cells in culture (96 well
format) to see if the
> antigen I am interested in is PIPLC sensitive or not (by cell surface
ELISA). Most buffers(HBSS,PBS)
> and/or media (HAMs F10, MEM) I can think of using to keep my cells going
during the digestion have
> between 100-150mM NaCL!
> Any ideas? Am I just reading an outdated paper?
> I would very much appreciate any help.
>  
> Sincerely,
> Galatia Politopoulou.

Reply:
I have used Bac. cereus PI-PLC from Boehringer to investigate a similar
problem, using removal of ecto-alkaline phosphatase as a positive control.
Worked out very nicely and using isoosmotic buffer conditions you can
still expect about 50% residual PI-PLC activity (Sunder et al,1978, J.
Biol. Chem. 253,4175-79). For details see in P. Jordan et al, 1992,
Biochem. Int. 26, 97-104. For further information hpeter at fm.ul.pt, good
luck



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