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Mol. Cells 2009; 27(3): 337-343

Published online March 31, 2009

https://doi.org/10.1007/s10059-009-0043-x

© The Korean Society for Molecular and Cellular Biology

Quantitative and Rapid Analysis of Transglutaminase Activity Using Protein Arrays in Mammalian Cells

Mi-Hye Kwon, Jae-Wan Jung, Se-Hui Jung, Jin-Young

Received: October 21, 2009; Revised: November 14, 2009; Accepted: January 7, 2009

Abstract

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutaminase activity in mammalian cells. Transglutaminases are a family of Ca2+-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N?-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the [3H]putrescine-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transglutaminase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

Keywords all-trans retinoic acid, on-chip activity assay, protein arrays, putrescine-incorporation assay, transglutaminase activity

Article

Research Article

Mol. Cells 2009; 27(3): 337-343

Published online March 31, 2009 https://doi.org/10.1007/s10059-009-0043-x

Copyright © The Korean Society for Molecular and Cellular Biology.

Quantitative and Rapid Analysis of Transglutaminase Activity Using Protein Arrays in Mammalian Cells

Mi-Hye Kwon, Jae-Wan Jung, Se-Hui Jung, Jin-Young

Received: October 21, 2009; Revised: November 14, 2009; Accepted: January 7, 2009

Abstract

We developed a novel on-chip activity assay using protein arrays for quantitative and rapid analysis of transglutaminase activity in mammalian cells. Transglutaminases are a family of Ca2+-dependent enzymes involved in cell regulation as well as human diseases such as neurodegenerative disorders, inflammatory diseases and tumor progression. We fabricated the protein arrays by immobilizing N,N?-dimethylcasein (a substrate) on the amine surface of the arrays. We initiated transamidating reaction on the protein arrays and determined the transglutaminase activity by analyzing the fluorescence intensity of biotinylated casein. The on-chip transglutaminase activity assay was proved to be much more sensitive than the [3H]putrescine-incorporation assay. We successfully applied the on-chip assay to a rapid and quantitative analysis of the transglutaminase activity in all-trans retinoic acid-treated NIH 3T3 and SH-SY5Y cells. In addition, the on-chip transglutaminase activity assay was sufficiently sensitive to determine the transglutaminase activity in eleven mammalian cell lines. Thus, this novel on-chip transglutaminase activity assay was confirmed to be a sensitive and high-throughput approach to investigating the roles of transglutaminase in cellular signaling, and, moreover, it is likely to have a strong potential for monitoring human diseases.

Keywords: all-trans retinoic acid, on-chip activity assay, protein arrays, putrescine-incorporation assay, transglutaminase activity

Mol. Cells
Jul 31, 2022 Vol.45 No.7, pp. 435~512
COVER PICTURE
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into mesodermal lineages like adipogenic, osteogenic, and chondrogenic. Alcian blue-positive extracellular matrix secreted by chondrocytes in the lacuna confirmed the chondrogenic differentiation of MSCs (Bashyal et al., pp. 479-494).

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