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
