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…

Offenlegungen

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

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Diesen Artikel zitieren
Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes. J. Vis. Exp. (Pending Publication), e21460, doi: (2023).

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