Janet B. Matsen:FLS pathway crude extract LCMS assay
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Steps & Comments
- ?? figure out how to make a table?
- acetonitrile --> denature enzymes to stop chemistry
- pivolate = internal standard
- pH for buffer chosen base on other metabolite measurement & assay protocols
- PMSF = phenol methylsulfonyl chloride: inhibits things that can break down proteins, supposedly. See Amanda's protein purification book.
- the percise OD you collect cultures at isn't critical.
- she centrifuges before ultracentrifuging because she wanted to compare crude extract (after regular centrifugation) to after ultracentrifugation.
- ultracentrifugation removes membrane proteins (which are NADH sinks) and desalting might remove formaldehyde sinks like glutathione.