Snapshot Blotting: Transfer of Nucleic Acids and Nucleoprotein Complexes from Electrophoresis Gels to Grids for Electron Microscopy

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 15; pp. 6870 - 6874
• Main Authors: Jett, Stephen D., Bear, David G.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 19-07-1994; National Acad Sciences; National Academy of Sciences
• Subjects: Biochemistry; Deoxyribonuclease EcoRI - ultrastructure; Dimers; DNA; DNA - chemistry; DNA - ultrastructure; Dyes; Electron microscopy; Electrophoresis; Electrophoresis, Agar Gel - methods; Gels; Microscopy, Electron; Nucleic acids; Nucleic Acids - chemistry; Nucleic Acids - ultrastructure; Nucleoproteins; Nucleoproteins - chemistry; Nucleoproteins - ultrastructure; Proteins; RNA; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.15.6870

We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy. The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay. To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation. Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions.