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Single-molecule motility assays and TIRF microscopy imaging

AL Andres Leschziner
KN Kendrick H Nguyen
S Samara Reck-Peterson
EK Eva Karasmanis

Last updated date: Oct 20, 2026 Views: 89 Forks: 0

original An abbreviated version of this protocol was published in Nature Communications
Cryo-EM captures early intermediate steps in dynein activation by LIS1
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Pre-made stock solutions 5XDLB with no KOAc

150mM HEPES pH7.4 with NaOH

10mM MgOAc 5mM EGTA

50% glycerol

 

5X TEV

50mM Tris.HCl pH8 750mM KCl

50% glycerol

 

5X GF150 Buffer

125mM HEPES pH7.4

750mM KCl

5mM MgCl2

 

 

300 nM Lis1 WT, del 298-308. 

Made MTs 488 LAST WEEK

 

Protein

Conc. Dim.

Buffer

dynein TMR from Aga K made on 01/27/2022- (A3-6) clear tubes

197 nMGF150 buffer: 25mM HEPES pH7.4, 150mM KCl, 1mM MgCl2, 1mM DTT, and 10% glycerol

Dynactin unlabeled

From 10/06/20 - Andrea's rack (B7-5)

190 nM

GF150 buffer: 25mM HEPES pH7.4, 150mM KCl, 1mM MgCl2, 1mM DTT, 0.1mM ATP and 10% glycerol

BicD2S-unlabeled John G's 07/17/2019 prep - (A2-11)

2 uMGF150 buffer: 25mM HEPES pH7.4, 150mM KCl, 1mM MgCl2, 1mM DTT, 10% glycerol

LIS1 WT- (AK/KN)

11.5 uMZ-TEV Buffer: 10 mM Tris pH 8, 2 mM MgAOc,150 mM KOAc, 1 mM EGTA,1mM DTT, 10%glycerol

LIS1 del 298-308 (2769)

10.1 uMZ-TEV Buffer: 10 mM TrispH 8, 2 mM MgAOc,150 mM KOAc, 1 mM EGTA,1mM DTT, 10%glycerol

 

Protein

Stock Con.

Desired Conc.

Desired Vol.

Add stock (uL)

Add buffer (uL)

Dynein*

197 nM

15 nM

13

1

12

Dynactin

190 nM

40 nM

7

2

8

BicD2S

2 uM

300 nM

10

1.5

8.5

Lis1 WT

11.5 uM

1.5 uM

23

3

20

LIS1 del 298-308

10.1 uM

1.5 uM

134.6

20

114.6

*Idiluted dynein to 5 nM: 10 uL GF150 + 5 uL of 15 nM dynein

 

TIRF buffers 

1 ml GF150 w/10% glycerol

ul

1 ml Z-TEV buffer

ul

2 mls DLBT

ul

1 mls DLBCT

ul

1X GF150 w/10% glycerol

998

5X Z-TEV stock

200

5X DLB (no salt)

400

5X DLB (no salt)

200

DTT (1M)

1

DTT (1M)

1

Taxol (10 mM)

4

Taxol (10 mM)

2

0.1mM ATP

1

ddH2O

799

DTT (1M)

2

DTT (1M)

1

 

 

 

 

ddH2O

1594

Casein (43 mg/ml)

24

 

 

 

 

 

 

ddH2O

757

*Idiluted dynein to 5 nM: 10 uL GF150 + 5 uL of 15 nM dynein TIRF buffers

 

Scavenger

ul

Energy

ul

DLBCT

48

DLBCT

42

gloxy

2

glucose

2

 

 

100 mM ATP

5

 

 

BME

1

 

 

 

 

TIRF motility assay 

MT is diluted in DLBT buffer. 488-Taxol MT made by KR on 12/24/21. Dilute 1:150. Dynein, dynactin and activator are diluted in GF150 buffer. Lis1 is diluted in Z-TEV buffer.

 

Final protein concentrations in the motility chamber:

 

Protein

Working stock

Final conc. (nM)

Dynein

12 nM

0.2

Dynactin

55 nM

0.92

BicD2S-

sfGFP

300 nM

5

Lis1 WT

1.5 uM

300

LIS1 E300D

1.5 uM

300

LIS1 del 298-

308

1.5 uM

300

 

TIRF Laser setting

100x 1x

Readout EMGain 17MHz at 16-bit EM Gain1-150 20% power in 561, 100ms exposure

 

Single molecule Motility Assay

 

  1. Make flow chambers with No. 1.5 coverslips and double-sided tape
  2. Flow 20 µl 1 mg/ml biotin-BSA into chambers, Wait 3 minutes
  3. Wash 2X with 20 µl DLBT
  4. Flow 20 µl 1 mg/ml streptavidin into chambers, Wait 3 minutes
  5. Wash 2X with 20 µl DLBT
  6. 3. Flow in appropriate dilution of MT in DLBT. Wait 3 minutes.
  7. Wash twice with 20uL of DLBCT.

Image all samples in duplicate in three fields of view for 3 minutes at 0.3s/frame triggered. Samples to image:

 

DDBS

  • 1uL of 5 nM dynein + 1uL of 55 nM dynactin + 1uL of 300 nM BicD2S: 10 min on ice
  • 1uL of the above DDB + 4uL ZTEV + 5uL DLBCT: 10 minutes on ice
  • Add 5uL S and 5uL E - image

 

DDBS Lis1 WT 300 nM

  • 1uL of 5 nM dynein + 1uL of 55 nM dynactin + 1uL of 300 nM BicD2S: 10 min on ice
  • 1uL of the above DDB + 4uL 1.5uM Lis1 + 5uL DLBCT: 10 minutes on ice
  • Add 5uL S and 5uL E - image

 

DDBS Lis1(del 298-308) 300 nM

  • 1uL of 5 nM dynein + 1uL of 55 nM dynactin + 1uL of 300 nM BicD2S: 10 min on ice
  • 1uL of the above DDB + 4uL 1.5uM Lis1(del 298-308) + 5uL DLBCT: 10 minutes on ice
  • Add 5uL S and 5uL E - image

Related files

pdf DDBs_Lis1_motility_Nguyenetal2025.pdf download
Copyright: © 2026 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Leschziner, A, Nguyen, K H, Reck-Peterson, S and Karasmanis, E(2026). Single-molecule motility assays and TIRF microscopy imaging. Bio-protocol Preprint. bio-protocol.org/prep3017.
  2. Nguyen, K. H. V., Karasmanis, E. P., Kendrick, A. A., Reck-Peterson, S. L. and Leschziner, A. E.(2025). Cryo-EM captures early intermediate steps in dynein activation by LIS1. Nature Communications 16. DOI: 10.1038/s41467-025-62185-z

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