Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates.
Biotechniques
; 30(4): 776-7, 780-1, 2001 Apr.
Article
en En
| MEDLINE
| ID: mdl-11314260
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 sensitivity of these assays.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Hemoglobinas
/
Beta-Galactosidasa
/
Genes Reporteros
/
Eritrocitos
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Biotechniques
Año:
2001
Tipo del documento:
Article
País de afiliación:
Estados Unidos