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 Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols. Segmented or polarity-marked microtubules can also be used to assess motor directionality.

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 Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols. Segmented or polarity-marked microtubules can also be used to assess motor directionality.

Recommended Products

Unlabeled Tubulin Protein

Labeled Tubulin Protein

CYCLED TUBULIN (Cat. No. 032005)

ALEXA 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)

Protocol

1. Construct Flow Cell

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.

Microtubule Gliding Assay - Construct Flow Cell

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.

Microtubule Gliding Assay - Coat with Motor Protein, Block with Pluronic, Add Microtubules

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.

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.

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] = 500 µg/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] = 500 µg/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

Recommended Products

Unlabeled Tubulin Protein

CYCLED TUBULIN (Cat. No. 032005)

Labeled Tubulin Protein

ALEXA 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)

Protocol

Step 1

1. Construct Flow Cell

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.

Steps 2-4

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.

Step 5

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.

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] = 500 µg/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] = 500 µg/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

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 Generating GMPCPP-Stabilized Microtubules or Generating Taxol-Stabilized Microtubules protocols. Segmented or polarity-marked microtubules can also be used to assess motor directionality.

Recommended Products

Unlabeled Tubulin Protein

CYCLED TUBULIN
(Cat. No. 032005)

Labeled Tubulin Protein

ALEXA 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)

Protocol

Step 1

1. Construct Flow Cell

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.

Steps 2-4

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.

Step 5

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.

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] = 500 µg/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] = 500 µg/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