[Protein-analysis] Re: how to make protein unfold curve

Frank Küster frank at kuesterei.ch
Mon Aug 29 09:21:10 EST 2005


"subberry" <zhangpy at gmail.com> wrote:

>  ^
>  |          oxoxoxox
>  |        o x
>  |     o  x
>  |    o  x
>  |   o  x
>  | xox x
>  +------------------------>
>           c(GdnHCl)/M
>
> In this diagram, "x" and "o"representing denature and renature process
> respectively, is it right?For "x" we can do it by dissolve the nature
> protein in the buffers with varying GdnHCl concentrations, and for "o"
> we can  denature the protein in high GdnHCl ,and Then dilute it into
> the buffers with varying
> GdnHCl concentrations, 

Exactly.  Sorry if I was unclear.

> So my question is how long can we measure after
> dissovling or dilute? is it we should wait the refolding (unfolding)
> reach equilibium?

Unfortunately the answer is: It depends.  Folding and unfolding kinetics
of different proteins reach from the millisecond time range to hours or
even days.  A really extreme case is my PhD-thesis-protein, I incubated
it for 3 months, and still the two "branches" (x and o) did not
completely meet; the culprit was slow unfolding in this case.

If you don't know anything about your protein, I would pippet the stuff
together one afternoon and measure the following day.  If both curves
don't coincide, keep the samples and measure again after two or three
days.  

If it takes longer than that, either you decide that you are interested
in the folding or unfolding kinetics of the protein, and systematically
investigate them, or otherwise you should consider to forget about
determining the thermodynamic stability.  Or you try different
denaturing methods - urea instead of guanidine hydrochloride could help,
or thermal denaturation - either in the CD or in a calorimeter.


Regards, Frank
-- 
In der Zeit, in der man einen defekten Riegel derartig seziert hat, dass man
verlässlich bestimmte Speicherbereich als "heile" garantieren(!) kann, kann 
man auch Pfandflaschen im Stadtpark sammeln, dass reicht dann für einen 
doppelt so großen neuen Riegel. ;)       [Jörg Rossdeutscher in d-u-g at l.d.o]


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