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."
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