Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes
Published: July 31, 2023
Abstract
Source: Stormberg, T., et al. Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp.(2019)
In this video, we demonstrate static atomic force microscopy to visualize assembled nucleosomes. This high-resolution imaging technique allows the study of the nucleosome structure.
Protocol
1. Functionalization of Mica Surface for Static AFM Imaging of Nucleosomes Prepare a 50 mM 1-(3-Aminopropyl) silatrane APS stock solution in deionized water as described. Store 1 mL aliquots of this solution at 4 °C until use. NOTE: The aliquots can be stored for more than a year at 4 °C. Prepare a working APS 1:300 solution for mica modification by dissolving 50 µL of the 50 mM APS stock in 15 mL dd H2O. NOTE:</strong…
Representative Results
Figure 1: Schematic of the process to prepare APS functionalized mica for AFM imaging of nucleosomes. (A) a piece of mica ~0.1 mm in thickness has both sides freshly cleaved. (B) The cleaved mica piece is promptly placed diagonally in a cuvette containing the APS solution and is set to incubate for 30 min. (C) Following the APS functionalization step, the APS-mica piece is transferred to a cuvette filled with dd…
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
| ageRuler 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 | |
| HEPES | Sigma-Aldrich, St. Louis, MO | H4034-25G | Catalog number for 25 g |
| Scotch Tape | Scotch-3M, St. Paul, MN | ||
| TESPA-V2 afm probe (for static imaging) | Bruker AFM Probes, Camarillo, CA | ||
| 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 | ||
| MultiMode Atomic Force Microscope | Bruker-Nano/Veeco, Santa Barbara, CA |
Etiketler
Bu Makaleden Alıntı Yapın
Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes. J. Vis. Exp. (Pending Publication), e21460, doi: (2023).