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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