Ammonium Acetate Protein Precipitation

Bill Melchior, NCTR/FDA WMELCHIOR at NTET.NCTR.FDA.GOV
Fri Jun 26 12:20:37 EST 1992


>In a BRL Focus (Vol 9, number 2), there was an article about parameters of
>ethanol precipitation using Ammonium Acetate.  In it, there was a description
>of a procedure to precipitate proteins from solutions containing protein and
>DNA using Ammonium Acetate.   In brief, the solution was made 2.5 M Ammonium
>Acetate, centrifuged15 minutes at 16000 x g, the supernatant transferred to a
>new tube, and the DNA precipitated by addition of 2 volumes Ethanol.

>Does anyone have any direct experience with this procedure?  Does it work?  

I first read about this in the 1985 BRL catalog, and routinely do it as part
of my cleanup of large plasmid preparations.  I found that the spectrum of
pBR322 did improve after precipitation from AmOAc:

 To DNA in low-salt buffer add 1/2 volume 7.5 M AmOAc.  Mix and chill.  
 Centrifuge if turbid; results depend on purity of preparation at this stage.
 Precipitate with twice the TOTAL volume of EtOH.  Wash DNA with 70% EtOH.

[comments about NaCl precipitation deleted]

>I'd like to see if I can eliminate Phenol/Chloroform extraction steps in
>preparation of templates for in vitro transcription in making antisense RNA
>probes.  Currently, I digest the cut plasmid with Proteinase K, then phenol
>extract twice, chloroform extract, then ethanol precipitate.  I'd like to see
>if I can Proteinase K digest, then salt out the protein with ammonium acetate
>or NaCl, followed by ethanol precipitation.  Is this a dumb idea?

I can't answer specifically, but (a) I'm not sure that AmOAc precipitation will
substitute for phenol; I'd be surprised if it worked with all types of proteins.
(b) I suspect that the peptides resulting from the proteinase digestion would 
be LESS likely to precipitate in AmOAC than the whole proteins.

I'm becoming more and more a fan of glassmilk purification of DNA, and it sounds
like this might be an ideal application.

Please let us know how whatever you try works.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators



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