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