Cargando…
A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these met...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226564/ https://www.ncbi.nlm.nih.gov/pubmed/22140457 http://dx.doi.org/10.1371/journal.pone.0027680 |
_version_ | 1782217641953329152 |
---|---|
author | Ehrnhoefer, Dagmar E. Skotte, Niels H. Savill, Jane Nguyen, Yen T. N. Ladha, Safia Cao, Li-Ping Dullaghan, Edie Hayden, Michael R. |
author_facet | Ehrnhoefer, Dagmar E. Skotte, Niels H. Savill, Jane Nguyen, Yen T. N. Ladha, Safia Cao, Li-Ping Dullaghan, Edie Hayden, Michael R. |
author_sort | Ehrnhoefer, Dagmar E. |
collection | PubMed |
description | Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders. |
format | Online Article Text |
id | pubmed-3226564 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32265642011-12-02 A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture Ehrnhoefer, Dagmar E. Skotte, Niels H. Savill, Jane Nguyen, Yen T. N. Ladha, Safia Cao, Li-Ping Dullaghan, Edie Hayden, Michael R. PLoS One Research Article Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders. Public Library of Science 2011-11-29 /pmc/articles/PMC3226564/ /pubmed/22140457 http://dx.doi.org/10.1371/journal.pone.0027680 Text en Ehrnhoefer et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Ehrnhoefer, Dagmar E. Skotte, Niels H. Savill, Jane Nguyen, Yen T. N. Ladha, Safia Cao, Li-Ping Dullaghan, Edie Hayden, Michael R. A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title | A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title_full | A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title_fullStr | A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title_full_unstemmed | A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title_short | A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture |
title_sort | quantitative method for the specific assessment of caspase-6 activity in cell culture |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226564/ https://www.ncbi.nlm.nih.gov/pubmed/22140457 http://dx.doi.org/10.1371/journal.pone.0027680 |
work_keys_str_mv | AT ehrnhoeferdagmare aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT skottenielsh aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT savilljane aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT nguyenyentn aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT ladhasafia aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT caoliping aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT dullaghanedie aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT haydenmichaelr aquantitativemethodforthespecificassessmentofcaspase6activityincellculture AT ehrnhoeferdagmare quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT skottenielsh quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT savilljane quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT nguyenyentn quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT ladhasafia quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT caoliping quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT dullaghanedie quantitativemethodforthespecificassessmentofcaspase6activityincellculture AT haydenmichaelr quantitativemethodforthespecificassessmentofcaspase6activityincellculture |