Huh7 and acetone;)
someone at microsoft.net
Sun Feb 8 11:27:02 EST 2004
"D.K." <dk at no.email.thankstospam.net> wrote in message
news:auib20lphth80n8ke5cavk6ld01fj0892d at 4ax.com...
> On Sun, 08 Feb 2004 00:13:09 -0500, "P.C." <PC at no.email.sorry> wrote:
> >acetone is a very harsh precipitant. I would expect almost any protein
> >to lose activity. It might work for very soluble, small proteins, may
> Sure, acetone is hard on many proteins. OTOH, it is surprisingly
> easy on many others. I'd guess the difference is just how
> well and compactly the protein is folded, and how much of a role
> an internal solvent plays.
> Most of the classic biochemistry in 1960s on fractionating
> yeast enzymes was done with acid or acetone precipitation.
> Acetone powder preparations are at the core of muscle contractile
> proteins biochemistry. And so on.
> That said, I wouldn't want any protein I worked hard to
> obtain to be exposed to acetone. Any acetone. The risk is just
> too high.
I was talking about E.coli alkaline phosphatase (AP). Not hard to get at
all: IPTG-inducible periplasmic expression, and further excreted into the
media. I am doing a 1960-style purification by adding up to 70% acetone to
the cleared culture broth, and get tons of protein. Luckily, the AP in the
media accounts for ~80% of detectable protein. I was just not very sure that
I should add Zn2+ (cofactor of AP)to the buffer (plain neutral Tris) I use
to reconstitute precipitated AP to perhaps recover more activity. I am
talking about mere ~30% of activity lost during precipitation.
Oh yeah, of course I probably would not use acetone on some hard to get
prots. Well, I might have, too, if it worked :-). Sometimes the simplest
thing is the easiest, and vice versa.
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