Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes

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…

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