Hoenger Lab
University of Colorado at Boulder
Department of Molecular, Cellular and Developmental Biology

Structural and Functional Investigations
into Cytoskeletal Assemblies by
Cryo Electron Microscopy
and 3D Image Analysis

 
Research:: Protocols and Procedures

Making Microtubules:

Polymerization Mix Protocol:

The microtubule polymerization mix consists of the following concetrations:

  • 80mM PIPES
  • 2mM GTP
  • 2mM MgCl2
  • 20 µM Taxol
  • pH 6.8 with KOH
  • 15% DMSO

Create the mix in the following order, follow the diagram at the left as a guide as to how to place the droplets of solution.

  1. 2µl GTP (100mM stock)
  2. 2µl Taxol (1mM stock)
  3. 4µl MgCl2 (100 mM stock)
  4. 77µl BRB80, add quickly to mix droplets
  5. 15µl DMSO, mix well
Mixing with Tubulin :

The polymerization mix is added 1:1 to Tubulin (10mg/ml) for a final tubulin concetration of 5mg/ml.

  1. Add 10µl to 10µl of 10mg/ml tubulin
  2. Incubate at 37ºC for 15-30 min.
  3. Store at room temperature.
  4. For best results wait 36 hours before freezing. However, microtubules are good for three days.
  5. If your objective is non-decorated microtubules, dilute 1:10 with BRB80.
Adding Motors :

For 10µl of solution (about 2 grids) so if more is needed, multiply as needed. Add 4 or 5µl to grid then freeze.

  1. 1µl AMP-PNP (10mM)
  2. 1µl microtubules (5mg/ml)
  3. 2µl motors (10mg/ml)
  4. 6µl BRB80

Solutions and Stocks:

BRB80:
  • 80mM PIPES
  • 1mM MgCl2
  • 1mM EGTA
  • pH 6.8 with KOH
Guanosine Triphosphate (GTP)
  • Diluted to 100mM in BRB80
AMP-PNP
  • Diluted to 10mM in BRB80

5′-Adenylylimidodiphosphate (AMP-PNP) is a
synthetically prepared non-hydrolyzable analog of
adenosine 5'-triphosphate.

McCl2
  • Diluted to 100mM in BRB80
Taxol
  • Diluted to 1mM in dimethyl sulfoxide (DMSO)
 

University of Colorado, MCDB, 347 UCB, Boulder, CO 80309-0347