May 23, 2022

Public workspaceLabeled microtubules for single-molecule imaging

DOI
dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1
Protocol Citation: John Salogiannis 2022. Labeled microtubules for single-molecule imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 19, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 46496
Keywords: single-molecule, microtubule, imaging, ASAPCRN
Abstract
This protocol is for making labeled taxol-stabilized microtubles to be used for single-molecule imaging assays by adhering to biotin slides.
Materials
Required Buffers:

5x BRB80:

  • Concentration400 millimolar (mM) PIPES, pH 6.8 with KOH
  • Concentration10 millimolar (mM) MgCl2
  • Concentration5 millimolar (mM) EGTA

2x Polymerization Mix:

  • Concentration2 x BRB80
  • Concentration2 millimolar (mM) DTT
  • Concentration2 millimolar (mM) GTP (Add last)
  • Concentration2 millimolar (mM) MgCl2 (Add second to last)
  • Concentration20 % DMSO
  • Mix well between adding each ingredient
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Please take notice of the buffer preparation in the Materials section
Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
50m
50m
In a prechilled 1.5 mL Eppendorf tube, make a Amount10 µL mixture of Concentration10 mg/mL tubulin .
The mixture should be 80% unlabeled, 10% biotin-tubulin, and 10% 405-tubulin.
Mix by gently flicking.
Mix
Let it sit TemperatureOn ice for Duration00:10:00 .
10m
Add equal volume of Concentration2 x polymerization buffer (Amount10 µL ). Mix by gently flicking.
Mix
Incubate at Temperature37 °C for Duration00:30:00 . Make a Concentration1 x BRB80 + Concentration1 millimolar (mM) DTT + Concentration20 micromolar (µM) Taxol stock and incubate at Temperature37 °C at the same time.
30m
Incubation
Add equal volume of prewarmed Concentration1 x BRB80 + DTT + Taxol (Amount20 µL ). Mix by gently flicking.
Mix
Incubate at Temperature37 °C for at least Duration00:10:00 (solution will be stable for hours at this point).
10m
Incubation
Store in the dark at TemperatureRoom temperature . Should be usable for several weeks, but more aggregates will appear over time.