In article <3dcq27$omj at apollo.it.luc.edu>, gnew at orion.it.luc.edu (Gerri Newfry) says:
>>Megan Oxberry (oxberry at numbat.murdoch.edu.au) wrote:
>: I am a PhD. student in Perth, Western Australia who is trying
>: desperately to purify tubulin for use in an assay to measure tubulin
>: polymerisation. So far I have tried using the polymerisation/depolymerisation method of
>: Shelanski et al. (1973) and the homogenisation, centrifugation and
>: dialysis method of Barrowman et al. (1984), both of which have been
>: unsuccessful.
>: Megan Oxberry oxberry at numbat.murdoch.edu.au
(Long responses by gerri...gnew at orion.it.luc.edu and
Ginnie Dress at biosci.arizona.edu)
I have tried to rigorously follow the procedure of Williams and Lee, preparation
of tubulin from brain, Methods in Enzymology 85:376-385, 1982. My only
changes are that a) I pH the buffer at 6.6, and b) I store my 1XMTP in 3 M
glycerol (PM3M buffer), and my 2XMTP in 0.4 M glycerol (PM0.4M buffer) at
-80 C. What the respondents are saying about brain freshness is right on
the money. When I obtain brains, I have the person removing the brain dip
the brain into ice water first, then place in crushed ice. I have used goat and
sheep brains as well as calf brains and the smaller animals' brains actually
have given me better MTP yields, presumably because their smaller brains
chill faster and thus retard tubulin degradation. But if I can't get the brains
homogenized within 1.5-2 hr after slaughter, yield and quality really drops off.
D. Murphy (Methods in Cell Biology 24:31-49, 1982) has graphed assembly vs
time and states: 'Ideally one should use brain tissue obtained within 20
min of slaughter'. One other trick that I've read about (but don't remember
where) may be to add a protease inhibitor like PMSF to the initial homogenate
to minimize tubulin decomposition. Good luck, SWM
mamber at synapse.bms.com
