I can't describe this one much better than the image I borrowed does. Membrane protein prep is a pain. Even if you are using some sort of a subcellular fractionation kit (and the ones I've used have not given me incredible membrane protein yields). This looks really promising, though and (cheap and) easy!
You can check out this method in JPR's ASAP section here
Sounds very promising. FASP only gives about 50% recovery. Enhanced FASP gives slightly higher yield. It would be interesting to see a head-to-head comparison.ReplyDelete
Is this method, and the ones that you refer to that give unsatisfactory protein yields, mainly for top down proteomics? Or, do you see this as being a possibly better approach for bottom up proteomics as well? (Related: are the bottom up membrane protein preps also unsatisfactory?) I ask because I'm new to proteomics and am intending to do LC-MS/MS for membrane proteins (in human).ReplyDelete
Here I'm definitely referring to bottom-up proteomics. I've never ever been happy with the coverage that I get from membrane proteins. My favorite methods are still limited loading into SDS-PAGE (let it run just until the proteins migrate in a little...say 60V for 10 minutes) or FASP, with the limitations that are mentioned in the comment above. A technique I've been dying to try for years is Josip Blonder's methanol based digestion method detailed in this paper, I think (http://onlinelibrary.wiley.com/doi/10.1002/pmic.200300543/abstract;jsessionid=90ED19340CF3552E104DE9FAD6E72C83.f02t02)Delete
They do this at Frederick National Labs all the time.