[3dem] Microtubule in vitro polymerisation

[Sindelar, Charles]

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


> Date: Thu, 9 Nov 2017 21:33:23 +0000
> From: Linda Sandblad <linda.sandblad at umu.se>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: [3dem] Microtubule in vitro polymerisation
> Message-ID: <C2BC2AAB-FD0A-46B5-B729-18B676F61784 at molbiol.umu.se>
> Content-Type: text/plain; charset="utf-8"
> 
> Hi!! I must mention, at the EM facility we have many beautiful samples, it?s just that I often think the artifacts are more interesting to discuss ;-)
> This is a question especially to all of you microtubule experts; When our students prepare mt from Cytoskeleton bovine tubulin it sometimes looks perfect, but sometimes it is all covered by those huge blobs, what do you think cause this artefact??
> Happy to hear your thoughts! Best Linda
> [cid:8D91DB92-CC19-4B6E-B5AF-9948CD785A79 at lan]
> 
> Linda Sandblad
> Department of Molecular Biology
> Ume? University
> Tel: +46 709324936