High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics
Published: July 31, 2023
Abstract
Source: Stumme-Diers, M. P., et al., Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp. (2019)
In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.
Protocol
1. Continuous Dilution Assembly of Mono-nucleosomes Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence. NOTE: To limit the unwanted formation of di-nucleosomes, each ‘arm' flanking the positioning sequence should not exceed ~150 bp. Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the…
Representative Results
Figure 1: Schematic of the syringe pump used for microscale nucleosome assembly. The assembly mixture is positioned to be in contact with the end of the syringe needle. As the dilution buffer is delivered by the syringe pump to the assembly mixture the concentration of NaCl is decreased, promoting nucleosome assembly. This figure is adapted from Stumme-Diers et al. <img alt="Fi…
開示
The authors have nothing to disclose.
Materials
| Plasmid pGEM3Z-601 | Addgene, Cambridge, MA | 26656 | |
| PCR Primers | IDT, Coralville, IA | Custom Order | (FP) 5'- CAGTGAATTGTAATACGACTC-3' (RP) 5'-ACAGCTATGACCATGATTAC-3' |
| DreamTaq polymerase | ThermoFischer Scientific, Waltham, MA | EP0701 | Catalog number for 200 units |
| PCR purification kit | Qiagen, Hilden, Germany | 28104 | Catalog number for 50 units |
| Tris base | Sigma-Aldrich, St. Louis, MO | 10708976001 | Catalog number for 250 g |
| EDTA | ThermoFischer Scientific, Waltham, MA | 15576028 | Catalog number for 500 g |
| (CENP-A/H4)2, recombinant human | EpiCypher, Durham, NC | 16-0010 | Catalog number for 50 ug |
| H2A/H2B, recombinant human | EpiCypher, Durham, NC | 15-0311 | Catalog number for 50 ug |
| H3 Octamer, recombinant human | EpiCypher, Durham, NC | 16-0001 | Catalog number for 50 ug |
| Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mL | ThermoFischer Scientific, Waltham, MA | 69574 | Catalog number for 10 devices |
| Sodium Chloride | Sigma-Aldrich, St. Louis, MO | S9888-500G | Catalog number for 500 mg |
| Amicon Ultra-0.5 mL Centrifugal Filters | Millipore-sigma, Burlington, MO | UFC501008 | Catalog number for 8 devices |
| HCl | Sigma-Aldrich, St. Louis, MO | 258148-25ML | Catalog number for 25 mL |
| Tricine | Sigma-Aldrich, St. Louis, MO | T0377-25G | Catalog number for 25 g |
| SDS | Sigma-Aldrich, St. Louis, MO | 11667289001 | Catalog number for 1 kg |
| Ammonium Persulfate (AmmPS) | Bio-Rad, Hercules, CA | 1610700 | Catalog number for 10 g |
| 30% Acrylamide/Bis Solution, 37.5:1 | Bio-Rad, Hercules, CA | 1610158 | Catalog number for 500 mL |
| TEMED | Bio-Rad, Hercules, CA | 1610800 | Catalog number for 5 mL |
| 4x Laemmli protein sample buffer for SDS-PAGE | Bio-Rad, Hercules, CA | 1610747 | Catalog number for 10 mL |
| 2-ME | Sigma-Aldrich, St. Louis, MO | M6250-10ML | Catalog number for 10 mL |
| PageRuler Prestained Protein Ladder | ThermoFischer Scientific, Waltham, MA | 26616 | Catalog number for 500 uL |
| Bio-Safe™ Coomassie Stain | Bio-Rad, Hercules, CA | 1610786 | Catalog number for 1 L |
| Nonwoven cleanroom wipes: TX604 TechniCloth | TexWipe, Kernersvile, NC | TX604 | |
| Muscovite Block Mica | AshevilleMica, Newport News, VA | Grade-1 | |
| Aminopropyl silatrane (APS) | Synthesized as described in 22 | ||
| HEPES | Sigma-Aldrich, St. Louis, MO | H4034-25G | Catalog number for 25 g |
| Scotch Tape | Scotch-3M, St. Paul, MN | ||
| Aron Alpha Industrial Krazy Glue | Toagosei America, West Jefferson, OH | AA480 | Catalog number for 2 g tube |
| MgCl2 | Sigma-Aldrich, St. Louis, MO | M8266-100G | Catalog number for 100 g |
| Millex-GP Filter, 0.22 µm | Sigma-Aldrich, St. Louis, MO | SLGP05010 | Catalog number for 10 devices |
| BL-AC10DS-A2 afm probe (for HS-AFM) | Olympus, Japan | ||
| Compound FG-3020C-20 | FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan | ||
| Compound FS-1010S135-0.5 | FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan | ||
| High-Speed Time-Lapse Atomic Force Microsocopy | Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan |
