Abstract
LacZ is widely used as a reporter in studies of gene expression patterns. β-galactosidase, the product of LacZ gene, is usually detected by X-gal/FeCN staining. In X-gal/FeCN staining, β-galactosidase catalyzes X-gal to produce blue precipitates, which indicate the expression patterns of the gene of interest. A newer LacZ detection method using S-gal/TNBT is more sensitive but plagued by high background. Here, we describe an improved procedure that combines advantageous steps from the two methods. By comparing with X-gal/FeCN and S-gal/TNBT methods in detecting the expression patterns of miR-322/503 and miR-451 at a series of developmental stages, the improved method showed higher sensitivity and lower background. Thus, the improved method could be an alternative way of β-galactosidase staining in low gene expression situations.
Highlights
IntroductionThe protein product of LacZ is β-galactosidase, which catalyzes certain substrates to produce visible precipitates for ready detection
LacZ is a common reporter gene used to study gene expression patterns [1, 2]
The most popular β-galactosidase substrate is X-gal. β-galactosidase catalyzes X-gal hydrolysis, giving rise to 5-bromo-4-chloro-3-hydroxyindole and galactose. 5-bromo4-chloro-3-hydroxyindole is oxidized into a dimer that forms blue precipitates in the presence of potassium ferricyanide and potassium ferrocyanide [3, 4]
Summary
The protein product of LacZ is β-galactosidase, which catalyzes certain substrates to produce visible precipitates for ready detection. The most popular β-galactosidase substrate is X-gal. Β-galactosidase catalyzes X-gal hydrolysis, giving rise to 5-bromo-4-chloro-3-hydroxyindole and galactose. 5-bromo4-chloro-3-hydroxyindole is oxidized into a dimer that forms blue precipitates in the presence of potassium ferricyanide and potassium ferrocyanide [3, 4]. The X-gal/FeCN method exerts high specificity and low background, it fails when β-galactosidase expresses at low levels [5, 6]. A new S-gal/TNBT method was proposed as an alternative in detecting low β-galactosidase expressions [5]. S-gal is another chromogenic substrate of β-galactosidase, showing higher sensitivity than X-gal when used together with FeCN [7]. The S-gal/ TNBT combination is much more sensitive than X-gal/FeCN, but has a severe overstaining
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