Hi Deb
The extraction buffer Jim Lawrence referred to was devised for running 2D gels, so
tributy phosphine was included because it is 1) a potent reducing agent and 2)
uncharged so it won't migrate off of the IEF gel during electrophoresis. If
you're not doing IEF, you can substitute TCEP (charged, less toxic, water soluble,
but with the same chemistry as TBP) or higher concentrations of DTT or (my last
choice) mercaptoethanol. If you are running 2D gels and want the benefits of TBP,
Bio-Rad sells a kit where it is already formulated and you only use a small
quantity at a time.
David
Deb McMillen wrote:
> Tributyl phosphine is pretty nasty stuff to deal with and dispose of--is
> there anything that could substitute for it?
>
> Deb McMillen
> Institute of Molecular Biology
> University of Oregon
> Eugene OR 97403
>
> On Thu, 14 Dec 2000, Lawrence, Jim wrote:
>
> > Jose,
> > Here's the best I've found. It is really, really good for extraction of
> > membrane proteins.
> > Taken from Molloy, Herbert, Walsh et al. 1998, Electrophoresis, vol. 19, pp.
> > 837-844
> > Listed as Sequential Extraction Solution #3:
> > 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 2 mM Tributyl phosphine and 40
> > Mm Tris-base, pH 9.5.
> >
> > Extract away and be impressed with how very little membrane pellet is left
> > after extraction with this solution.
> >
> > Jim Lawrence
> > Pioneer Hi-Bred Intl.
> > 7300 NW 62nd Ave
> > Johnston, IA 50131
> > 515-270-5909
> >
> > Keep your head up and your stick down.
> >
> >
> > > -----Original Message-----
> > > From: Jose Cesar Rosa [mailto:jrosa@cisunix.unh.edu]
> > > Sent: Wednesday, December 13, 2000 4:48 PM
> > > To: Recipients of ABRF List
> > > Cc: abrf@aecom.yu.edu
> > > Subject: Re: CHAPS for membrane protein extraction
> > >
> > >
> > > Hi Bob,
> > > Thanks for your reply and so many questions. I am using the
> > > Tris-HCl Buffer
> > > with CHAPS and others components listed below to extract as
> > > many membrane
> > > proteins as possible and also others proteins after cell
> > > lysis for glycan
> > > characterization. My special attention is for glycoproteins
> > > and many of
> > > them are integral proteins. So, I am not interested in any particular
> > > membrane protein, I only want to have a rich fraction in
> > > glycoproteins (but
> > > do not want to use lectins for that). I have been used this solution
> > > containing CHAPS (above of CMC, is strange too, but it is
> > > described in many
> > > protocols for solubility of proteins for 2D gel analysis). In
> > > short, my
> > > special request is if someone have been evaluated CHAPS
> > > (include different
> > > concentrations from cmc (about 0.4%) to above that or any
> > > other detergent
> > > whose main concerning is to have a rich membrane protein
> > > fraction in solution.
> > > Thanks a lot
> > > Cesar
> > >
> > >
> > > At 11:16 AM 12/13/00 -0500, you wrote:
> > > >Hi Jose,
> > > >
> > > > You need to be a little more specific with your
> > > question as there is
> > > >no single detergent that optimally extracts all proteins from all
> > > >membranes. Some more specific questions you might want to be asking
> > > >yourself are: (a) are you aiming to extract a specific (or a few)
> > > >protein(s)/enzyme(s), or as many membrane proteins as
> > > possible? (b) If it's
> > > >a single protein/enzyme extraction - is this a novel
> > > purification your lab
> > > >is working on, or are there data in the literature
> > > describing conditions
> > > >for its extraction/purification (if the latter, check them
> > > out!)? If it's
> > > >a novel purification, only you can empirically determine
> > > which detergent(s)
> > > >best extracts your protein(s) of interest. How efficiently
> > > CHAPS will
> > > >solubilize your protein(s) is, again, something you yourself need to
> > > >assess. Maybe try some non-ionic or anionic/cationic
> > > detergents? (c)
> > > >Finally, what is it you want to do with your extracted
> > > proteins once you
> > > >get them out of the membranes? Further purification? 2-D
> > > gel analysis?
> > > >Do you need to maintain enzyme activity? Will you need to remove the
> > > >detergent, etc?
> > > >
> > > >If this "CHAPS buffer" you refer to is a standard extraction solution
> > > >commonly used in the type of work you're doing, I confess
> > > I'm not familiar
> > > >with it. At 4% CHAPS (w/v I'm assuming), we're looking at
> > > ~65 mM detergent
> > > >(monomer MW = 615) which is well above CHAPS' cmc (6-10 mM
> > > in 0-50 mM Na+)
> > > >- meaning it is at a sufficiently high concentration to
> > > solubilize integral
> > > >membrane proteins & lipids. Have you looked at the ABRF document at
> > > >http://
> > > www.abrf.org/ABRFNews/1997/December1997/dec97detergent.html? I'm
> > > >not sure if CHAPS is discussed in it. I hope some of this
> > > has been helpful.
> > > >
> > > >Bob Keefe
> > > >
> > > >
> > > >At 05:30 PM 12/12/2000 -0800, you wrote:
> > > > >Dear ABRFers.
> > > > >Is someone out there has an idea how much efficient is
> > > CHAPS buffer (35mM
> > > > >Tris-HCl pH 8.0, 8M urea, 65mM DTT and 4% CHAPS) for
> > > membrane protein
> > > > >extraction? Is there other detergent which could be more
> > > efficient than
> > > >CHAPS?
> > > > >Thanks in advance for any information on this matter.
> > > >
> > > > >Best regards,
> > > > >Cesar
> > > > >Jose Cesar Rosa PhD
> > > > >Dept. of Chemistry
> > > > >University of New Hampshire
> > > >
> > > >
> > > >
> > > >Robert G. Keefe, Ph.D.
> > > >Wadsworth Center/NYS Dept. of Health
> > > >Genomics Core Facility
> > >
> > > Jose Cesar Rosa PhD
> > > Dept. of Chemistry
> > > University of New Hampshire
> > > 23 College rd. Parsons Hall
> > > Durham NH 03824
> > > phone: (603) 862-3797
> > >
> >
This archive was generated by hypermail 2b29 : Fri Jan 05 2001 - 08:42:46 EST