Related Experiment Videos
Immuno-atomic force microscopy of purple membrane
D J Müller1, C A Schoenenberger, G Büldt
1M. E. Müller Institute for Microscopic Structural Biology, Biozentrum University of Basel, Switzerland.
Biophysical Journal
|April 1, 1996
Summary
Atomic force microscopy visualizes biological structures using antibodies for surface identification. This technique reveals bacteriorhodopsin topography by selectively removing and reattaching antibodies during imaging.
Area of Science:
- Biophysics
- Microscopy
- Structural Biology
Background:
- Atomic force microscopy (AFM) enables high-resolution imaging of native biological structures.
- Conventional immunolabeling techniques are adapted for AFM to identify specific molecular surfaces.
- Bacteriorhodopsin in purple membranes serves as a model system for surface topography studies.
Purpose of the Study:
- To utilize antibodies targeting the C-terminus of bacteriorhodopsin for distinguishing membrane surfaces during AFM imaging.
- To reveal the molecular topography of the cytoplasmic and extracellular surfaces of purple membranes.
- To investigate the dynamic labeling and relabeling capabilities of antibodies in buffer solution.
Main Methods:
- Employing atomic force microscopy (AFM) for imaging purple membranes in a buffer solution.
- Using antibodies against the C-terminus of bacteriorhodopsin for surface-specific labeling.
- Applying controlled forces (>= 0.8 nN) with the AFM scanning stylus to remove antibodies and expose the underlying topography.
- Observing antibody relabeling upon stylus retraction.
- Conducting immuno-dot blot analysis and SDS-PAGE to confirm antibody specificity.
Main Results:
- AFM successfully imaged the extracellular surface of purple membrane at 0.7 nm resolution, showing distinct protrusions per bacteriorhodopsin monomer.
- Antibody removal by the scanning stylus allowed visualization of the cytoplasmic surface topography.
- The scanned membrane area was observed to relabel with antibodies within 10 minutes after stylus retraction.
- Cleavage of the bacteriorhodopsin C-terminus by papain abolished antibody labeling, confirming specificity.
Conclusions:
- Antibody-based AFM imaging provides a high-resolution method for distinguishing and visualizing different surfaces of biological membranes.
- The dynamic labeling and removal of antibodies allow for sequential surface topography mapping.
- This approach offers a powerful tool for studying the structure and organization of membrane proteins like bacteriorhodopsin.