A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.
Anal Biochem
; 484: 75-81, 2015 Sep 01.
Article
en En
| MEDLINE
| ID: mdl-26026386
ABSTRACT
We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Colorimetría
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Metaloproteinasa 2 de la Matriz
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Proteínas ADAM
/
Pruebas de Enzimas
/
Proteínas de la Membrana
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2015
Tipo del documento:
Article