[3dem] Microtubule in vitro polymerisation

[Sindelar, Charles]

One other critical piece of advice is to not let your taxol get too old- I’m not sure how widely appreciated this is. We find that frozen aliquots of taxol in DMSO go bad after no more than 6 months of storage at -80°C.  After this point, polymerized microtubules get progressively junkier and eventually fail to polymerize at all, as we have learned the hard way from multiple repetitions [!]

Cheers, Chuck

Hi Linda,
Regarding the blobs you sometimes get on your microtubules:  It could be
aggregated tubulin that couldn't polymerize.  Do you spin the tubulin at
high speed (100k x g, 10min in table-top ultracentrifuge)  before
polymerizing?  Also, we normally put the tubulin through at least one
additional polymerization/depolymerization cycle before freezing away in
small aliquots for use in experiments.

--
Michelle Moritz, PhD
Agard and Krogan Labs
UCSF MC2240, Rm S416
Dept. of Biochemistry & Biophysics
600 16th Street
San Francisco, CA 94158-2517
415-502-2930

Hi Linda, we have often seen imperfections in our microtubules when using the Cytoskeleton proteins (historically, certain batches seemed to be worse than others).  One possibility is that the artifacts (we saw ?junk?, not sure if I would describe as ?blobs?) are due to inactivated tubulin, but regardless of the source - the microtubules cleaned up almost perfectly in our hands by passing them through a glycerol cushion (i.e. spin them down through 70% glycerol plus buffer at, i.e., ~50K RPM in a Beckman tabletop ultracentrifuge TLA 120.2 rotor, wash pellet, and resuspend)

Hope that helps - Chuck

Hi Linda-

We would also see those sorts of blobs from time to time in our negative stain images of microtubules.  Is batch-to-batch variation in tubulin the only thing that is changing between experiments?  We would sometimes include small amounts of NP-40 detergent in our buffers, the stock of which would oxidize with time after being opened and left in the fridge.  Sometimes swapping for a new bottle would solve this type of issue.  Not sure if this is helpful in your case, but non-tubulin buffer variation may be another thing to consider.

Good luck!
-Greg Alushin

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