Hi Theresa, To put Bert's comment in different words, ultracentrifugation is another purification step. As much as ultracentrifugation needs additional time and resuspending membrane is a real pain, I find that it really does separate my membrane protein from a lot of other soluble E.coli contaminants, proteolytic products, etc. Also, if I skipped ultracentrifugation and simply solubilized the supernatant from the cell lysate (I guess that's what you meant by "whole cell"), I worry that my yields might be compromised because of incomplete solubilization of all of the membrane protein in whatever volume and percent of detergent I experimented with.
Many protocols recommend a second step of ultracentrifugation to get rid of *unsolubilized junk* after the first ultracentrifugation, membrane resuspension and detergent solubilization steps. I skip the second round of ultracentrifugation for one of my membrane proteins because the pellet from the second round is invisible and the protein is pure and functional after purification. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Mon, Jan 6, 2014 at 5:03 PM, Bert Van-Den-Berg < bert.van-den-b...@newcastle.ac.uk> wrote: > No real need, some folks do it like that. > However, you'll need to fish out your protein from more junk and you may > have more proteolytic degradation. > Also, you will need more detergent compared to "membrane-only" extraction, > especially when using mild detergents. > > bert > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Theresa > Hsu [theresah...@live.com] > Sent: Monday, January 06, 2014 9:56 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Membrane fractionation > > Dear crystallographers > > When we purify membrane proteins, we often fractionate the E. coli cell > membrane by ultracentrifugation. Is there a need for this step instead of > solubilizing the whole cell with detergents? > > Thank you. > > Theresa >
