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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

DOI: 10.3791/3061-v
What you'll learn
  • ✓ Prepare plasmid DNA and protein samples for AFM visualization
  • ✓ Operate tapping mode AFM and calibrate imaging parameters
  • ✓ Acquire and interpret AFM images of DNA-protein complexes
  • ✓ Distinguish between near-physiologic and alternative sample preparation methods
Protocol

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Difficulty
advanced
Total time
~4-6 hours per sample set (including AFM equilibration, probe mounting, and multi-image acquisition)
Biosafety
BSL-1

Steps

1
Prepare DNA and protein samples and mount AFM probe

Prepare plasmid DNA and cytoplasmic protein samples through biochemical manipulation. Mount the AFM probe according to manufacturer specifications for subsequent imaging.

▶ 01:12
2
Prepare AFM instrument and establish imaging conditions

Calibrate the tapping mode AFM, establish near-physiologic buffer conditions, and optimize instrumental parameters for biomolecule visualization.

▶ 02:43
3
Image mica surface and biomolecules of interest

Scan the mica sample surface to locate DNA and protein complexes, acquire high-resolution AFM images at appropriate scan rates and amplitudes.

▶ 04:33
4
Analyze and interpret representative AFM images

Review acquired AFM images to visualize plasmid DNA, protein structures, and DNA-protein interaction sites with analysis of topographical features.

▶ 06:25
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