beta galactosidase assay
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).
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)
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