Labeled microtubules for single-molecule imaging
John Salogiannis
Abstract
This protocol is for making labeled taxol-stabilized microtubles to be used for single-molecule imaging assays by adhering to biotin slides.
Before start
Please take notice of the buffer preparation in the Materials section
Steps
Create taxol-stabilized labeled microtubules (can be reused for multiple weeks):
In a prechilled 1.5 mL Eppendorf tube, make a 10µL mixture of 10mg/mL.
The mixture should be 80% unlabeled, 10% biotin-tubulin, and 10% 405-tubulin.
Mix by gently flicking.
Let it sit On ice for 0h 10m 0s.
Add equal volume of 2x (10µL). Mix by gently flicking.
Incubate at 37°C for 0h 30m 0s. Make a 1x + 1millimolar (mM) + 20micromolar (µM) stock and incubate at 37°C at the same time.
Add equal volume of prewarmed 1x + DTT + Taxol (20µL). Mix by gently flicking.
Incubate at 37°C for at least 0h 10m 0s (solution will be stable for hours at this point).
Store in the dark at Room temperature. Should be usable for several weeks, but more aggregates will appear over time.
