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Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes
Published on: February 9, 2009
The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with
A Lönnroth1, U Skoglund, K T Tokuyasu
1Department of Molecular Genetics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Journal of Microscopy
|May 1, 1993
Summary
Balbiani ring granules, essential premessenger ribonucleoprotein (RNP) particles, were visualized using electron microscope tomography. Embedding in polyvinyl alcohol (PVA) with uranyl acetate staining offers a rapid, effective method for preserving their structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Electron Microscopy
Background:
- Balbiani ring granules are premessenger ribonucleoprotein (RNP) particles crucial in gene expression in dipterans.
- Understanding the ultrastructure of these RNP particles is key to deciphering their function.
Purpose of the Study:
- To biochemically isolate and visualize Balbiani ring granules in three dimensions.
- To evaluate different preparation methods for electron microscopy of isolated RNP particles.
- To determine an optimal method for preserving RNP particle structure for tomography.
Main Methods:
- Biochemical isolation of Balbiani ring granules from Chironomus tentans.
- Preparation of RNP particles using positive staining, negative staining, and adsorption staining (polyvinyl alcohol embedding with uranyl acetate).
- Three-dimensional visualization using electron microscope tomography and comparison with in situ samples.
Main Results:
- Positive staining resulted in compacted, deformed particles with lost internal structure.
- Negative and adsorption staining preserved outer contours and central cavities.
- Adsorption staining in polyvinyl alcohol (PVA) revealed the internal 7-nm elementary fiber, though less distinctly than in plastic-embedded samples.
- PVA-embedded particles showed sufficient stability for electron microscope tomography.
Conclusions:
- Embedding in PVA with uranyl acetate is a rapid and effective method for preserving and staining isolated RNP particles.
- This PVA embedding technique facilitates detailed structural analysis of RNP particles via electron microscope tomography.
- The method allows for comprehensive tilt-series acquisition, crucial for high-resolution 3D reconstruction.
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