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Microtubule Gliding Assay
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.
Protocols
1. Construct Flow Cells
Assemble a glass “flow-cell” chamber by sandwiching two strips of double stick tape between a glass slide and glass coverslip. The two tape strips should be placed ~10 mm apart to create a chamber that holds ~15 µl volume. The chamber is open on either end so that buffer can be flowed through the chamber by pipetting into one end while simultaneously wicking from the other end.
Upon starting the experiment, do not allow the chamber to dry out. Keep the volume saturated at all times.
2. Coat with Motor Protein
Infuse the protein of interest into the flow cell until the entire volume is filled (~15 µl). Incubate for 3 minutes.
Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.
3. Block with Pluronic
Infuse 20 µl of 1% Pluronic F-127. Incubate for 1 minute.
Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.
4. Add Microtubules
Infuse 20 µl of polymerized microtubules. Incubate for 3 minutes.
Wash the flow cell by wicking 60 µl of Flow Cell Buffer through the chamber.
NOTE :
The optimal motor protein and microtubule concentrations vary, but are typically in the 1 µM and 0.2 µM range, respectively. Motor proteins and polymerized microtubules can typically be diluted in Wash Buffer if necessary.
Flow Cell Buffer is used as the final wash because it contains the oxygen scavenging mix required for imaging. It is necessary to make fresh Flow Cell Buffer every couple of hours to maintain optimal performance of the Oxygen Scavenging Mix.
The optimal motor protein and microtubule concentrations vary, but are typically in the 1 µM and 0.2 µM range, respectively. Motor proteins and polymerized microtubules can typically be diluted in Wash Buffer if necessary.
Flow Cell Buffer is used as the final wash because it contains the oxygen scavenging mix required for imaging. It is necessary to make fresh Flow Cell Buffer every couple of hours to maintain optimal performance of the Oxygen Scavenging Mix.
5. Image
Begin imaging the flow cell immediately. The optimal time interval varies depending on the protein of interest, but is typically in the 2-5 second range.
Use neutral density filters and fresh Oxygen Scavenging Mix to attenuate photobleaching.
Recommended Products
Unlabeled Actin Protein
Buffer
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