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
> >
>
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