BIOL309 Study Guide - Final Guide: Electric Eel, Affinity Chromatography, Tetrodotoxin
8 Jun 2017
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The first voltage-gated sodium channel genes were isolated from the electric organ of the electric eel. Tetrodotoxin (ttx) is a potent neurotoxin that binds to sodium channels. a) Electric eel organ constitutes 60-70% of the body length. Can deliver 650v/1a- enough to stun a horse: a binding assay based on high specificity of neurotoxins. Tetrodotoxin ttx (a potent neurotoxin isolated from puffer fish) Step1- purification of sodium channels: given the fact that tetrodotoxin (ttx) binds specifically to the sodium channels, they were purified by affinity chromatography: tetrodotoxin immobilized on sepharose beads was used as an affinity resin. The rabbit antiserum was obtained for later use (screening expression libraries) Ii) aliquot #2: was subjected to trypsin (protease) digestion, the peptides obtained post trypsin digestion were separated by reverse phase-high performance. Liquid chromatography (rp-hplc): the sequence of the peptides was determined by edman"s degradation, degenerate radiolabeled (p32) synthetic probes were synthesized based on peptide sequences.
