beta galactosidase assay

Henk Veldman H.Veldman-2 at neuro.azu.nl
Mon Oct 8 04:17:03 EST 2001



Wolfgang Schechinger schreef:

> Dear all,
>
> could anyone give me some hints on the chemistry behind that assay for
> beta galactosidase? It's for using beta gal as reporter gene assay.
>
> the transfected cells are fixed with formaldehyde/glutardialdehude for 5 min
> nad then washed with PBS (there is no further permeabilization)
>
> and then incubated with a solution containing
>
> X-Gal (of course)
> magnesium chloride
> potassium hexacyanoferrates (equimolar amounts of FeII and FeIII
> hexacyanoferrate) in water.
>
> any ideas?
>

The X-Gal substrate is split by the enzyme galactosidase in its constituents:
galactosidase and 5-bromo-4-chloro-3-indoxyl. Under the right redox conditions
(created by the hexacyanoferrates) the latter will convert to insoluble blue
dimers (in fact a derivative of the indigo that originally was used to dye blue
denim). Other indoxyl compounds are used in similar indigogenic methods to
localize other enzyms (esterases, phosphatases).

Henk
--
H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)





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