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Novel method to detect beta-galactosidase by a dot-blotting assay on nitrocellulose membrane using 6-bromo-2-naphthyl beta-D-galactopyranoside as substrate

Wan, L.; Van Huystee, R.B.

Journal of Agricultural and Food Chemistry 42(11): 2499-2501

1994


ISSN/ISBN: 0021-8561
DOI: 10.1021/jf00047a024
Accession: 009110498

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A novel method to detect beta-galactosidase by dot-blotting samples on nitrocellulose membrane and staining the membrane with azo dye coupling technique using 6-bromo-2-naphthyl beta-D-galactopyranoside as substrate is presented. The enzymatic reaction to split the substrate is pH-dependent, and the sensitivity of this method could be increased by more than 10-fold with an additional treatment of 0.01 M sodium carbonate after the coupling of diazo-blue B with 6-bromo-2-naphthyl released by beta-galactosidase. As low as 0.0015 unit of beta-galactosidase could be detected within 10 min. This improved beta-galactosidase staining method may be used for a native or IEF gel to show the beta-galactosidase active band and may also detect other hydrolases using substrates for the azo dye coupling technique.

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