Microtubule Gliding Assay

Microtubule gliding assay graphical abstract

Overview

The motility of kinesin and/or dynein ensembles can be assessed in microtubule gliding assays wherein glass imaging chambers, or flow cells, are coated with motor proteins and infused with microtubule-containing solutions. For a guide to polymerizing fluorescent and/or biotinylated microtubules, visit the protocols Generating GMPCPP-Stabilized Microtubules and Generating Taxol-Stabilized Microtubules. Segmented or polarity-marked microtubules can also be used to assess motor directionality.

Protocol

Recommended Products

Unlabeled Tubulin Protein

Cycled Tubulin Protein Product ImageCycled Tubulin
(Cat. No. 032005)

Labeled Tubulin Protein

Cy5 Labeled Tubulin Protein Product ImageAlexa Fluor® 488
(Cat. No. 048805)

Alexa Fluor® 594
(Cat. No. 059405)

Alexa Fluor® 647
(Cat. No. 064705)

Cy5
(Cat. No. 065005)

Biotin - XX
(Cat. No. 033305)

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