If I were to have a putative gene sequence, how would I determine
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If I were to have a putative gene sequence, how would I determine its protein and likely function by laboratory techniques?
Here's what I'm currently thinking:
>Isolate the specific gene of interest
>Amplify the DNA sequence
>Insert into vector and transform into an organism
>Isolate the expressed recombinant protein
>Isolate the proteins by size & MW (SDS-PAGE then MW Chromatography?)
>Determine AA sequence of protein by Western blot (?)
>DNA Microarray to determine localisation & expression of that gene sequence
>Then finally gene knockout in a species to observe function & functional gene
how about:
>wait until college
Use a computer to look for open reading frames.
>>7781370
it would be easiest to order the gene from a synthetic lab, and place a terminal 6x his tag (or any sort of affinity tag, but with a proteolytic sequence in between to cleave tag after all is said and done). then express in organism, purify via nickel column, cleave tag, then characterize protein
>>7781370
also, you cannot determine aa sequence from wb analysis noob. you might just do repetitive edman degradation
>>7781370
X-ray crystallography
>>7781370
Fuck doing it experimentally. Sequence the fucker and then BLAST it.