in-vivo cross-linking?

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Apr 12 09:34:29 EST 2002



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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