Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.
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 sensitivity of these assays.
Nazarenko
D A
DA
University of Rochester School of Medicine, Rochester, NY, USA.
Dertinger
S D
SD
Gasiewicz
T A
TA
D000818:Animals; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism*; D017930:Genes, Reporter; D006454:Hemoglobins / Q000378:metabolism*; D007763:Lac Operon; D008163:Luminescent Measurements; D008967:Molecular Biology / Q000379:methods; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D006454:Hemoglobins; D001616:beta-Galactosidase