Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography.
Anal Biochem
; 291(1): 27-33, 2001 Apr 01.
Article
en En
| MEDLINE
| ID: mdl-11262153
ABSTRACT
The currently available methods for the detection of proteases in tissue sections are characterized by limited substrate specificity and low sensitivity and are also cumbersome. We have developed a novel in situ zymography method that uses a synthetic substrate conjugated to a fluorescent tag for detection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulting in an approximately 100-fold increase in the fluorescent signal. In order to minimize the diffusion of the fluorescent tag, the substrate is incorporated into 1% agarose prior to overlaying onto the tissue section. This method retains the morphological details of the tissue section, is highly sensitive and specific for the designated peptide sequence, and provides information regarding the functional status of the enzyme. Thus, this method could be used for detection and monitoring of enzymatic activity in tissue sections for a variety of applications.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cicatrización de Heridas
/
Heridas Penetrantes
/
Elastasa Pancreática
/
Metaloproteinasa 9 de la Matriz
/
Metaloproteinasa 1 de la Matriz
/
Colorantes Fluorescentes
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Anal Biochem
Año:
2001
Tipo del documento:
Article
País de afiliación:
Estados Unidos