1a) Noda and Numa in 1984 isolated the first voltage-gated sodium channel gene from the electric organ of the electric eel. Electric organs contain a high density of sodium channels. Tetrodotoxin (TTX) is a potent neurotoxin that blocks sodium channels. No sodium channel gene had been identified by this time in 1984. The only clue that these researchers had, was that tetrodotoxin was a highly selective toxin for sodium channels.
a) Explain the approach that Noda and Numa would use to separate and purify the sodium channel protein from all the other electric eel organ proteins.
b) Explain the approach could they use to identify that the sodium channel protein was 280 kDa (or 280,000 MW) (5 marks)
1b) Once Noda and Numa had a purified sodium channel protein, they digested and sequence some of the amino acid sequence by Edman Degradation technique. From the purified sodium channel protein and/or the amino acid sequence, Noda and Numa could generate a probe to find the cDNA sequence that codes for the sodium channel.
a) Explain how they generated the type of probe that they could use to screen a library by hybridization.
b) Explain the steps in creating the library that would be used for screening for the sodium channel gene. Name the type of vector used for the library, tissue source for making the library, what they cloned into the multiple cloning site of the vector and the type of library that they generated (5 marks)