in-vivo cross-linking?

Emir Khatipov khatipovNO at NOuchicago.edu
Fri Apr 12 17:13:49 EST 2002


Frank, Thank you. I finally got an answer the question on the difference
between the PFA and FA I was asking around for some time. Still, a bit
strance why would some people fix yeast for example in FA overnight...
Emir



"Frank O. Fackelmayer" <Frank at Fackelmayer.de> wrote in message
news:3CB6F076.6013905 at Fackelmayer.de...
>
>
> Emir Khatipov wrote:
> >
> > Intracellular protein concentration of protein is >100mg/ml, and other
> > proteins will be crosslinked to your complex of interest.
>
> not necessarily. But of course, you need good controls, as always...
>
> >
> > I for your question, I would try paraformaldehyde or formaldehyde. These
> > chemicals are generally used to fix (in fact - crosslink "everything to
> > everything") yeast and mammalian cells. PFA (usually 1.5-4% solution in
PBS,
> > sometimes supplemented with ~1% sucrose) works faster - within a few
minutes
> > (<5min). Formaldehyde requires longer times of incubation
(30min-overnight).
>
> no, formaldehyde is chemically the same as paraformaldehyd in solution.
> In fact, dissolved PFA and FA work equally well and fast, but for some
> experiments it is better to use "paraformaldehyde" than formaldehyde:
> commercial liquid formaldehyde (37%) is stabilized with methanol
> (usually 5-10%), whereas "paraformaldeyde" is dry, chemically pure
> formaldehyde that can be dissolved in the buffer of your choice. The
> "stabilization" by methanol may adversely affect your experiment.
>
>
> Frank





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