Help with tubulin purification

Virginia Dress Dress at biosci.arizona.edu
Thu Dec 22 19:28:54 EST 1994


In article <3dcq27$omj at apollo.it.luc.edu>, gnew at orion.it.luc.edu (Gerri
Newfry) wrote:
> 
> 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.
(protocol deleted)
> 		gerri...gnew at orion.it.luc.edu

OK, first thing, MES is one of the Good's buffers (like Tris).  Now back to
the
original question.  I'm pretty sure Shelanski used calf brains, I've
haven't
seen many protocols that use lamb brains.  You do need to use calf brains,
and not the brains from older cattle, the yeild of tubulin drops
preciptously after a certain age.  That may or may not be true of lambs
also, you may want to check on that.  Better yet are pig brains, doesn't
make any difference how old they are, you still get a good yeild.  A good
yield is 1mg purified tubulin (2 cycles of
polymerization/depolymerization)/ g wet weight of brain.
		Polymerization/depolymerization purification does NOT work well with some

protozoa (yeast, and others like Tetrahymena).  It may have to do with
their complement of MAPs.  So I'm not sure you'll be able to use that sort
of protocol.  The reagents listed above are basically the stuff I used
(that's info off the top of my head, I can look it up if need be).  I
homogenized in a 
Waring blender, be careful not to homogenize too long, you'll start to get
some
stringy tangled material and that is bad.  Keep everything cold, cold,
cold.
The faster you can get the brain out, get it chilled, remove the meninges
(loaded with proteases because of the high degree of vascularization), and 
homogenize and do the first spin, the better.  All protein purification
protocols are best done as quickly as possible, so I would discourage the 
overnight incubations Gerri mentions. (at least until after you get it to
work once).
		How are you assaying for whether or not your purification worked?  I was
using
turbidity upon polymerization.  Do you have any ideas where you are losing
the
protein?  Try doing a relatively small prep and take samples at every
stage.
		So a short summary of a windy commentary:
					1.  Chill brain as fast as possible and keep cold.
					2.  Check into the age of lamb vs. yield or try pig brains
				 3.  Remove the meninges as completely as possible without nicking
brain
											tissue.
					4.  Keep the purification moving along as quickly as possible
					5.  Don't homogenize the brain tissue too long in the blender
					6.  Depolymerization/polymerization may not work for protozoa (or
Ascaris)

If you have more specific questions feel free to get in touch.

Ginnie
Dress at biosci.arizona.edu				   



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