Phenol/Ether Protein ppt : Phase Inversion?

H.V.Taylor harold.taylor at uni-tuebingen.de
Wed Dec 6 05:53:32 EST 2000


mlysik at hsc.vcu.edu wrote:

>Hi all, maybe someone can help me out a bit.  I've been trying to concentrate some dilute
>protein solutions for SDS-PAGE and have been testing different methods which I've seen
>referenced in this newsgroup.  So far, I've tried the MeOH/Chloroform extraction, which 
>seems to work ok except that I need to use 1.5mL tubes, which makes the process too 
>time consuming (starting with 11 1-mL samples). The phenol/ether extraction looks to be
>moire ideal since the whole volume stays below 1.5 mL.  
Dear Melanie,

as to phenol/ether extraction I'd agree with Peter Pediaditakis' reply
and would dilute the salt. However, after trying various extraction /
precipitation methods for proteins I find that the MeOH/CHCl3 method
gives the most representative results (as compared to say an original
muscle cytosol sample) and to is scalable to large samples. 

You'll discover that each method has it's drawbacks and will have to
decide which is the least problematic for your application. I've
listed a few references below: TCA (1), MeOH/CHCl3 (2), Silica gel
(3), Coomassie G-250 (4) and Pyrogallol red (5). Of these, IMHO TCA is
the worst and should be avoided. The other four give similar results
the differences being speed and quality: (3) and (5) are quite fast -
about 20 min. - (4) allows you to simply utilize the Bradford protein
assay samples (and adjust to SDS PAGE samples to comparable amounts of
protein) while (2) give to closest to a 1:1 representation of all of
proteins within the sample.

This turned out longer than I'd intended but I hope it helps you
anyway,

Harold

References
1. Peterson, E.A. and Greenberg, D.M. (1952) J.Biol.Chem. 194,
359-375.
2. Wessel-D and Flugge-UI (1984)   Anal.Biochem 138, 141-143.
3. Maresh, G.A. and Dunbar, B.S. (1988)   Electrophoresis 9, 54-57.
4. Marshall T and Williams KM (1992)   Electrophoresis 13, 887-888.
5. Aguilar RM et al. (1999)   Anal Biochem 267, 344-350.






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