Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes

Overview

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

1. 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.
2. 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: This working solution can be stored at room temperature for several days.
3. Cut 1 x 3 cm strips of mica from high-quality mica sheets (see Table of Materials for mica used here).
   1. Check that the piece fits when placed diagonally in a cuvette. Use the tip of sharp tweezers, a razor blade, or scotch tape, to cleave layers of the mica until both sides are freshly cleaved and the piece is as thin as ~0.1 mm (Figure 1A). Immediately place the mica piece into the APS-filled cuvette and incubate for 30 min (Figure 1B).
4. Transfer the mica piece to a cuvette filled with dd H2O and soak for 30 s (Figure 1C). Completely dry both sides of the APS-mica strip under an argon flow.
   NOTE: A non-woven cellulose and polyester cleanroom wipe (recommended wipe detailed in materials) can be used to aid in wicking water from the edge of the mica when drying.
5. Use the dry mica strip now for the sample preparation. Otherwise, store the piece in a clean, dry cuvette (Figure 1D).
   NOTE: Additional storage in a vacuum for 1-2 h is recommended when the environment is humid. The protocol can be paused here.

2. Preparation of Nucleosome Samples on APS-Mica for Static AFM Imaging

1. Apply double-faced adhesive tape to several magnetic pucks and place them to the side.
2. Cut the APS-mica substrate to the desired size (1 x 1 cm squares for the MM AFM instrument used here). Place these pieces in a clean petri dish and keep them covered.
3. Prepare three dilutions of the assembled nucleosomes (final nucleosome concentrations of 0.5, 1.0, and 2.0 nM) using a 0.22 µm filtered buffer containing 10 mM HEPES pH 7.5 and 4 mM MgCl₂.
   NOTE: To limit the loss of nucleosomes at the low final concentration, the dilutions should be done one at a time, immediately prior to deposition on the APS-mica.
4. Deposit 5-10 µL of the diluted nucleosome sample at the center of the APS-mica piece, and let it incubate for two minutes. Gently rinse the sample with 2-3 mL of dd H₂O to remove all buffer components. After each ~0.5 mL of dd H₂O is used, gently shake the mica to remove the excess rinse water.
   NOTE: A disposable syringe is recommended for this rinsing step.
5. Dry the deposited sample under a light flow of clean argon gas.
   NOTE: The sample is now ready to be imaged or can be stored in a vacuum cabinet or desiccator filled with argon. Samples prepared and stored as described have been imaged one year following preparation with no quality loss. The protocol can be paused here.

3. Static AFM Imaging of Nucleosomes

1. Mount an AFM tip on the tip holder. Use a tip that has a spring constant of ~40 N/m and a resonance frequency between 300 and 340 kHz (see the Table of Materials for the cantilevers used here).
2. Mount the sample prepared in section 3 on the AFM stage being careful not to contact the sample surface.
3. Position the laser over the cantilever until the sum is at the maximum and adjust the vertical and lateral deflection values to near zero.
4. Tune the AFM probe to find its resonance frequency and adjust the drive amplitude and set the image size to 100 x 100 nm. Click the engage button to begin the approach.
5. Once approached, gradually optimize the Amplitude Setpoint until the surface of the sample is clearly seen. Increase the scan size to 1 x 1 µm and the resolution to 512 x 512 pixels. Click the capture button followed by the engage button to begin image acquisition.
   NOTE: The images in Figure 2 show the smooth background that can be expected when imaging these samples.

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 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.

Materials

Name	Company	Catalog Number	Comments
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