Adhering Microtubules to Flow Cells

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 generating fluorescent, biotinylated microtubules, please see the “Generating GMPCPP-Stabilized Microtubules” protocol.

Recommended Products


Unlabeled Tubulin


Labeled Tubulin


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.

2. Coat with Biotin BSA

Infuse biotinylated BSA at 2 mg/ml into the flow cell until the entire volume is filled (~15 µl). Incubate for 10 minutes.

Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.


3. Add Streptavidin

Infuse 20 µl of streptavidin at 10 mg/ml. Incubate for 10 minutes.

Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.


4. 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.


5. Add Microtubules

Infuse 20 µl of fluorescent, biotinylated GMPCPP microtubules at 100 ng/ml [tubulin]. Incubate for 20 minutes. GMPCPP microtubules can be diluted in 1X Tubulin PEM Buffer if necessary.

Wash the flow cell by wicking 60 µl of Flow Cell Buffer through the chamber.


 NOTE :

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.

6. Image

Dilute the protein of interest to desired concentration in Flow Cell Buffer. Infuse into flow cell and begin imaging immediately.

Use neutral density filters and fresh Oxygen Scavenging Mix to attenuate photobleaching.

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 generating fluorescent, biotinylated microtubules, please see the “Generating GMPCPP-Stabilized Microtubules” protocol.

Recommended Products


Unlabeled Tubulin

Unlabeled Tubulin

Labeled Tubulin

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 2-5

2. Coat with Biotin BSA

Infuse biotinylated BSA at 2 mg/ml into the flow cell until the entire volume is filled (~15 µl). Incubate for 10 minutes.

Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.


3. Add Streptavidin

Infuse 20 µl of streptavidin at 10 mg/ml. Incubate for 10 minutes.

Wash the flow cell by wicking 60 µl of Wash Buffer through the chamber.


4. 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.


5. Add Microtubules

Infuse 20 µl of fluorescent, biotinylated GMPCPP microtubules at 100 ng/ml [tubulin]. Incubate for 20 minutes. GMPCPP microtubules can be diluted in 1X Tubulin PEM Buffer if necessary.

Wash the flow cell by wicking 60 µl of Flow Cell Buffer through the chamber.

 NOTE :

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 6

6. Image

Dilute the protein of interest to desired concentration in Flow Cell Buffer. Infuse into flow cell and begin imaging immediately.

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

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