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.
1. Functionalization of Mica Surface for Static AFM Imaging of Nucleosomes
2. Preparation of Nucleosome Samples on APS-Mica for Static AFM Imaging
3. Static AFM Imaging of Nucleosomes
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 H2O for a 30 s rinse. (D) The APS-mica piece is stored in a cuvette until use.
Figure 2: Example AFM images of H3 and CNEP-A nucleosomes. (A) Sample image of H3 mono-nucleosomes deposited on APS-mica, captured using static AFM. Each bright blob is a nucleosome core particle with the flanking DNA regions appearing as noodle-like arms. The long noodle-like features are free DNA particles that are not associated with a histone core. For this image, a 2 nM nucleosome concentration was used, providing a uniform distribution across the surface, with little to no crowding. (B) This nucleosome sample was deposited at 1 nM and is much less populated than the 2 nM used in (A). This demonstrates the direct effect that nucleosome dilution has on the surface density of nucleosomes.
The authors have nothing to disclose.
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 |