Hello, as the question says above I would like to know how you go from a cell line to obtaining the structure. I will be using sf9 insect cells.
From what I understand so far I will have to infect these cells with a Baculovirus that I harvest and amplify. The insect cells themselves then go into large scale expression and once they are express the proteins it has to be purified through chromatography? At what stage do the detergents come into play? After the chromatography can the protein be used for vapour diffusion to create crystals which then go through x-ray crystallisation?
Im very new to structural work so just being able to understand the process and timeline better would be a great help thank you!
As Ali Mahmoudpour suggested, start reading if you're new. Here are some articles you could start with. Good luck!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580798/
http://www.sciencedirect.com/science/article/pii/S0005273613002447
http://www.nature.com/nmeth/journal/v6/n1/full/nmeth.f.240.html
http://www.nature.com/nmeth/journal/v7/n6/full/nmeth0610-429.html
http://www.cell.com/trends/biochemical-sciences/fulltext/S0968-0004(07)00084-9
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075773/
http://sbkb.org/article/141
Membrane proteins are notoriously difficult to crystallize. That's where detergents come into play. Detergents will keep the membrane proteins in solution, because crystallization is possible only for proteins in solution, not those that are aggregated, which membrane proteins usually become in aqueous solution because they have large hydrophobic domains. BTW I would follow the advice of the previous poster, Ali Mahmoudpour -- you need to start reading everything you can get your hands on about protein structure, crystallization and diffraction. Research Gate is useful, but it cannot make you a protein biochemist.
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