Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy.
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.
Jett
S D
SD
Department of Cell Biology, University of New Mexico School of Medicine, Albuquerque 87131.
Bear
D G
DG
D004247:DNA / Q000737:chemistry / Q000648:ultrastructure; D015246:Deoxyribonuclease EcoRI / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D008854:Microscopy, Electron; D009696:Nucleic Acids / Q000737:chemistry / Q000648:ultrastructure*; D009698:Nucleoproteins / Q000737:chemistry / Q000648:ultrastructure*; D014158:Transcription, Genetic
D009696:Nucleic Acids; D009698:Nucleoproteins; D004247:DNA; D015246:Deoxyribonuclease EcoRI