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
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