Method Article

High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

July 8th, 2025

In This Article

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Source: Stumme-Diers, M. P., et al., Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp. (2019)

In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Continuous Dilution Assembly of Mono-nucleosomes

  1. Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.
    NOTE: To limit the unwanted formation of di-nucleosomes, each ‘arm' flanking the positioning sequence should not exceed ~150 bp.
    1. Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the 423 bp substrate with 122 and 154 bp arm lengths used here, use forward (5'-CAGTGAATTGTAATACGACTC-3') and reverse (5'-ACAGCTATGACCATGATTAC-3') primers.
    2. Add tubes containing....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Laboratory syringe pump for precise liquid handling in chromatography and PCR experiments.
Figure 1: Schematic of the syringe pump used for microscale nucleosome assembly. The assembly mixture is positioned to be in contact with the end of the syringe needle. As the dilution buffer is delivered by the syringe pump to the assembly mixture the concentration of NaCl is decreased, promoti.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Plasmid pGEM3Z-601Addgene, Cambridge, MA26656
PCR PrimersIDT, Coralville, IACustom Order(FP) 5'- CAGTGAATTGTAATACGACTC-3'<br/> (RP) 5'-ACAGCTATGACCATGATTAC-3'
DreamTaq polymeraseThermoFischer Scientific, Waltham, MAEP0701Catalog number for 200 units
PCR purification kitQiagen, Hilden, Germany&nbsp;28104Catalog number for 50 units
Tris baseSigma-Aldrich, St. Louis, MO10708976001Catalog number for 250 g
EDTAThermoFischer Scientific, Waltham, MA15576028Catalog number for 500 g
(CENP-A/H4)2, recombinant humanEpiCypher, Durham, NC16-0010Catalog number for 50 ug
H2A/H2B, recombinant humanEpiCypher, Durham, NC15-0311Catalog number for 50 ug
H3 Octamer, recombinant humanEpiCypher, Durham, NC16-0001Catalog number for 50 ug
Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mLThermoFischer Scientific, Waltham, MA69574Catalog number for 10 devices
Sodium ChlorideSigma-Aldrich, St. Louis, MOS9888-500GCatalog number for 500 mg
Amicon Ultra-0.5&nbsp;mL Centrifugal Filters&nbsp;Millipore-sigma, Burlington, MOUFC501008Catalog number for 8 devices
HClSigma-Aldrich, St. Louis, MO258148-25MLCatalog number for 25 mL
TricineSigma-Aldrich, St. Louis, MOT0377-25GCatalog number for 25 g
SDSSigma-Aldrich, St. Louis, MO11667289001Catalog number for 1 kg
Ammonium Persulfate (AmmPS)&nbsp;Bio-Rad, Hercules, CA1610700Catalog number for 10 g
30% Acrylamide/Bis Solution, 37.5:1Bio-Rad, Hercules, CA1610158Catalog number for 500 mL
TEMEDBio-Rad, Hercules, CA1610800Catalog number for 5 mL
4x Laemmli protein sample buffer for SDS-PAGEBio-Rad, Hercules, CA1610747Catalog number for 10 mL
2-MESigma-Aldrich, St. Louis, MOM6250-10MLCatalog number for 10 mL
PageRuler Prestained Protein Ladder&nbsp;ThermoFischer Scientific, Waltham, MA26616Catalog number for 500 uL
Bio-Safe&trade; Coomassie StainBio-Rad, Hercules, CA1610786Catalog number for 1 L
Nonwoven cleanroom wipes: TX604 TechniCloth&nbsp;TexWipe, Kernersvile, NCTX604
Muscovite Block MicaAshevilleMica, Newport News, VAGrade-1
Aminopropyl silatrane (APS)Synthesized as described in 22
HEPESSigma-Aldrich, St. Louis, MOH4034-25GCatalog number for 25 g
Scotch TapeScotch-3M, St. Paul, MN
Aron Alpha Industrial Krazy GlueToagosei America, West Jefferson, OHAA480Catalog number for 2 g tube
MgCl2Sigma-Aldrich, St. Louis, MOM8266-100GCatalog number for 100 g
Millex-GP Filter, 0.22&nbsp;&micro;mSigma-Aldrich, St. Louis, MOSLGP05010Catalog number for 10 devices
BL-AC10DS-A2 afm probe (for HS-AFM)Olympus, Japan
Compound FG-3020C-20&nbsp;FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan&nbsp;
Compound FS-1010S135-0.5&nbsp;FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan&nbsp;
High-Speed Time-Lapse Atomic Force MicrosocopyToshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

DNA UnwrappingHigh Speed AFMMica SubstrateNucleosome ImagingCantilever DeflectionHistone CoreNucleosome HeightTime Lapse Imaging

Related Articles