Monday, November 4, 2019
Easy & Fast Subcellular Fractionation -- All MS-compatible reagents!
I haven't done a subcellular fractionation in a while, but I've got a project coming my way probably since global didn't do a good job of grabbing the nuclear protein(s) of interest.
Most (all?) of the subcellular fractionation techniques were designed for something else. Maybe they were for getting nuclear DNA or for electron microscopy of mitochondria. They weren't designed for LCMS, and you can tell because of how much time is necessary to clean out the large list of non-LCMS compatible compounds.
This great new paper (published ASAP, so probably not indexed by your library yet) at JPR couldn't come at a better time. THIS is my new protocol.
Nothing going into this is incompatible with LCMS. No gross detergents or salts (or sugars...) to remove by solid phase extraction (SPE) or in-gel digestion. A protocol designed for LCMS analysis of subcellular fractions.
The efficiency increase (compared to anything I've ever tried) is dramatic. They start with as little as e5(!!) cells and pull out phosphorylation sites(!!) from subcellular fractions!