The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins
Published: April 30, 2023
Abstract
Source: Tripathi, A., et al. Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays. J. Vis. Exp. (2021).
In this video, we perform an in vitro motility assay to analyze the actin-myosin interactions. The gliding movement of actin filaments on immobilized myosin molecules is visualized using fluorescence microscopy.
Protocol
1. Gliding actin filament assay Coverslip preparation Make a 1% nitrocellulose solution in amyl acetate. Obtain a tissue culture dish (150 x 25 mm) and add a circular filter paper (125 mm diameter) to the bottom of the dish. Load eight No. 1.5 thickness 22 mm square coverslips onto a rack and wash with approximately 2-5 mL of 200-proof ethanol followed by 2-5 mL of distilled water (dH2O). Repeat this washing step, ending with water. Then, dry the c…
Representative Results
Figure 1: Preparation of functionalized flow-cell chambers. (A) Begin with a cleaned microscope slide, two pieces of double-sided tape cut to approximately 2 cm, and a functionalized coverslip. (B) Add the tape to the center of the microscope slide. (C) Attach the coverslip to the tape with the coating (i.e., nitrocellulose) facing down and gentl…
Divulgazioni
The authors have nothing to disclose.
Materials
| Amyl Acetate | Ladd Research Industries | 10825 | |
| ATP | Millipore Sigma | A7699 | |
| Bovine Serum Albumin | Millipore Sigma | 5470 | |
| Calmodulin | PMID: 2985564 | ||
| Catalase | Millipore Sigma | C40 | |
| Circular Filter Paper – Gliding Assay | Millipore Sigma | WHA1001125 | |
| Coverslip Rack | Millipore Sigma | Z688568-1EA | |
| Coverslips: Gliding Acting Filament Assay | VWR International | 48366-227 | |
| DL-Dithiothreitol | Millipore Sigma | D0632 | |
| Double-Sided Tape | Office Depot | 909955 | |
| EGTA | Millipore Sigma | E4378 | |
| Ethanol | Fischer Scientific | A4094 | |
| G-actin | PMID: 4254541 | G-actin stock can be stored at 200 μM in liquid N2. | |
| Glucose | Millipore Sigma | G8270 | |
| Glucose Oxidase | Millipore Sigma | G2133 | |
| KCl | Fischer Scientific | P217-500 | |
| Large-Orifice Pipet Tips | Fischer Scientific | 02-707-134 | |
| Methylcellulose | Millipore Sigma | M0512 | |
| Microscope Slides | Fischer Scientific | 12-553-10 | |
| MOPS | Fischer Scientific | BP308-100 | |
| Nitrocellulose | Ladd Research Industries | 10800 | |
| Razor Blades | Office Depot | 397492 | |
| Rhodamine-Phalloidin | ThermoFisher Scientific | R415 | Stock can be diluted in 100% methanol to a final concentration of 200 μM. |
| Tissue Culture Dish – Gliding Assay | Corning | 353025 | Each tissue culture dish can hold approximately nine coverslips. |
| Smooth-sided 200 µL Pipette Tips | Thomas Scientific | 1158U38 | |
| EQUIPMENT | |||
| Centrifuge | ThermoFisher Scientific | 75006590 | |
| Microscope | Nikon | Model: Eclipse Ti with H-TIRF system with 100x TIRF Objective (N.A. 1.49) | |
| Microscope Camera | Andor | Model: iXon DU888 EMCCD camera (1024 x 1024 sensor format) | |
| Microscope Environmental Control Box | Tokai HIT | Custom Thermobox | |
| Microscope Laser Unit | Nikon | LU-n4 four laser unit with solid state lasers for 405nm, 488nm, 561nm, and 640nm | |
| Optima Max-Xp Tabletop Ultracentrifuge | Beckman Coulter | 393315 |
Tags
Citazione di questo articolo
The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins. J. Vis. Exp. (Pending Publication), e21215, doi: (2023).