Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates

BioTechniques
D A NazarenkoT A Gasiewicz

Abstract

beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sen...Continue Reading

Citations

Oct 13, 2006·Toxicological Sciences : an Official Journal of the Society of Toxicology·Jeffrey C BemisThomas A Gasiewicz
Oct 11, 2012·Bacteriophage·David A SchofieldCaroline Westwater
Nov 26, 2015·Proceedings of the National Academy of Sciences of the United States of America·Shimyn SlomovicJames J Collins
May 23, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Wayne F Patton

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