A quantitative method for the specific assessment of caspase-6 activity in cell culture
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- A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture
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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.
Original language | English |
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Journal | PLOS ONE |
Volume | 6 |
Issue number | 11 |
Pages (from-to) | e27680 |
ISSN | 1932-6203 |
DOIs | |
Publication status | Published - 2011 |
- Amino Acid Sequence, Animals, COS Cells, Caspase 6, Cell Culture Techniques, Cell Extracts, Cercopithecus aethiops, Enzyme Assays, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Humans, Kinetics, Lamins, Luminescence, Mice, Molecular Sequence Data, Neurons, Peptides, Substrate Specificity
Research areas
ID: 153451326