BIPN140 Lecture 6

BIPN140 Lecture 6 - Lecture 6 2010 BIPN140 October 12,...

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Lecture 6 October 12, 2010 BIPN140 Reading: Chapter 4 Structure and Function of Ion Channels What do channels look like? •visualization: EM, X-ray crystallography; acetylcholine receptors (AChR); K channels •need to isolate from membrane •purification using ligands: AChR from Torpedo electric organ- BgTX; Na and Ca channels from mammalian striated muscle (TTX, STX-Na channels; dihydropyridines-Ca channels) •sodium channel subunits, electrophoretic gels: α 260kDa; β 1, 36kDa; β 2, 33kDa Primary structure (amino acid sequence) - from the gene N-(amino) terminal analysis •terminal sequencing of purified protein •manufacture of degenerate oligonucleotides •manufacture of cDNA library from tissue of interest •probe library with radiolabeled oligonucleotide •isolate clones; find overlapping clones to cover full length of the gene •sequence the clones •conceptual translation back into amino acids Homology screening (probing) and sequencing Expression cloning and sequencing
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BIPN140 Lecture 6 - Lecture 6 2010 BIPN140 October 12,...

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