Adhering Microtubules to Flow Cells

Adhering microtubules to flow cell graphical abstract

Overview

The motility of single kinesin and/or dynein molecules can be assessed by a TIRF-based assay wherein biotinylated microtubules are affixed to glass imaging chambers, or flow cells, followed by infusion with fluorescent motor proteins. For a guide to polymerizing fluorescent, biotinylated microtubules, visit the Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols. Segmented or polarity-marked microtubules can also be used to assess motor directionality.

Protocol

Buffers

Wash Buffer

  • 694 µl H20 | final volume = 1000 µl
  • 200 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 1 µl MgCl2 @ 1 M | [final] = 1 mM
  • 5 µl ATP @ 200 mM | [final] = 1 mM
  • 100 µl casein @ 5 mg/ml | [final] = 0.5 mg/ml

Flow Cell Buffer

  • 297 µl H20 | final volume = 500 µl
  • 100 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 0.5 µl MgCl2@ 1 M | [final] = 1 mM
  • 2.5 µl ATP @ 200 mM | [final] = 1 mM
  • 50 µl casein @ 5 mg/ml | [final] = 0.5 mg/ml
  • 50 µl Oxygen Scavenging Mix @ 10X | [final] = 1X

10X Oxygen Scavenging Mix

  • 30 µl Tubulin PEM Buffer @ 1X | final volume = 50 µl
  • 5 µl glucose oxidase @ 20 mg/ml | [final] = 2 mg/ml
  • 5 µl catalase @ 3.5 mg/ml | [final] = 0.35 mg/ml
  • 5 µl beta-mercaptoethanol @ 50% | [final] = 5%
  • 5 µl glucose @ 450 mg/ml | [final] = 45 mg/ml