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. Ultimately I would like to purify protozoal parasite
: tubulin but for now I have been using lamb brain and nematode (Ascaris)
: intestine as it is easier to obtain large amounts of material.
: 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.
: Has anyone any suggestions?
I can tell you our protocol, but I'm not sure it's different from those
you mention above, since it's not ME that actually uses this protocol.
We use calf brains.
MT Buffer:
0.1 M MES, pH 6.4, 4oC (it doesn't say what MES is, but if you don't
know either, I will ask--no one is here right now)
1 mM EGTA
1 mM GTP (or 0.1 mM GTP and 2.5 mM ATP)
0.5 mM MgCl2
Brain needs to be freshl Place it in a slurry of ice ASAP following
removal from animal.
Homogenize brain in 1 ml of 0.1 MES buffer for each gram of tissue, on ice.
Centrifuge at 45 K g for 2.5 h at 4oC
Remove supernatant and make it 30% glycerol. Warm to 37oC for at least
45 min (to allow MT polymerization). Meanwhile, run warm water over teh
rotor to break the cold and warm it to 25oC, by putting it into a warm
incubator.
Centrifuge at 45 K g for 2.5 h at 25oC
Supernatant is discarded (save an aliquot for the gel). Pellets (first
cycle MT) are resuspened in 0.2 ml MT buffer per gram of brain (original
tissue weight). Loosen pellets from the side of the tube with a spatula,
add a little buffer and pour the pellet in buffer into a new tube. Use a
small polytron tip to homogenize the pellet about 5 seconds, then
continue homogenizing in a small glass on glass homogenizer. For a rough
prep, one can stop here. Store MTs at 4oC for short term, or at -20oC
for longer term. Run sample on a small gel to check the putirty. If
more pure MTs are desired, continue....with each cycle, putiry goes up,
but yield goes down.
Chill MT's on ice at least 45 min (or overnight)
Centrifuge at 75 K g for 75 min at 4oC
Discard pellets. Make supernatant 30 % glycerol. Incubate at 37oC at
least 45 min (Warm the rotor)
Centrifuge at 75 K g for 75 min at 25oC
Pellets should contain purified MT protein (second cycle). Resuspend in
0.1 M MES buffer, store at -20oC
: Hopefully,
: Megan Oxberry oxberry at numbat.murdoch.edu.au
Good luck, I hope this helped.
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gerri...gnew at orion.it.luc.edu
