Home › Analytical Chem › Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy
Analytical Chem JoVE (Open Access) Citable · DOI

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

DOI: 10.3791/57902-v
What you'll learn
  • ✓ Monitor real-time assembly and disassembly of anthrax toxin complexes using biolayer interferometry
  • ✓ Release and visualize macromolecular protein assemblies via electron microscopy
  • ✓ Identify protein complex components using mass spectrometry analysis
Protocol

Biopharma Insights Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

Difficulty
advanced
Total time
~4–6 hours per sample (BLI monitoring + EM prep + MS analysis)
Biosafety
BSL-2

Steps

1
Assemble prepore anthrax toxin on biosensor

Load PDEA-modified amine reactive biosensor surface with anthrax toxin components to monitor real-time prepore complex formation using biolayer interferometry.

▶ 01:28
2
Assemble pore anthrax toxin on biosensor

Continue BLI monitoring as prepore converts to pore-form anthrax toxin complex on the same biosensor surface.

▶ 04:37
3
Release and identify macromolecular assemblies

Remove assembled complexes from BLI biosensor surface and analyze using negative-stain electron microscopy for visualization and mass spectrometry for component identification.

▶ 05:26
4
Analyze dynamic complex data

Integrate BLI kinetic data with electron microscopy structural images and mass spectrometry results to characterize assembly dynamics and complex composition.

▶ 06:50
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