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Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies
Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Mic...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Portland Press Ltd.
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682752/ https://www.ncbi.nlm.nih.gov/pubmed/23560750 http://dx.doi.org/10.1042/BJ20121742 |
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author | Davies, Paul Hinkle, Kelly M. Sukar, Nour N. Sepulveda, Bryan Mesias, Roxana Serrano, Geidy Alessi, Dario R. Beach, Thomas G. Benson, Deanna L. White, Charles L. Cowell, Rita M. Das, Sonal S. West, Andrew B. Melrose, Heather L. |
author_facet | Davies, Paul Hinkle, Kelly M. Sukar, Nour N. Sepulveda, Bryan Mesias, Roxana Serrano, Geidy Alessi, Dario R. Beach, Thomas G. Benson, Deanna L. White, Charles L. Cowell, Rita M. Das, Sonal S. West, Andrew B. Melrose, Heather L. |
author_sort | Davies, Paul |
collection | PubMed |
description | Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease. |
format | Online Article Text |
id | pubmed-3682752 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Portland Press Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-36827522013-06-27 Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies Davies, Paul Hinkle, Kelly M. Sukar, Nour N. Sepulveda, Bryan Mesias, Roxana Serrano, Geidy Alessi, Dario R. Beach, Thomas G. Benson, Deanna L. White, Charles L. Cowell, Rita M. Das, Sonal S. West, Andrew B. Melrose, Heather L. Biochem J Research Article Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease. Portland Press Ltd. 2013-06-13 2013-07-01 /pmc/articles/PMC3682752/ /pubmed/23560750 http://dx.doi.org/10.1042/BJ20121742 Text en © 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/3.0/ 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 work is properly cited. |
spellingShingle | Research Article Davies, Paul Hinkle, Kelly M. Sukar, Nour N. Sepulveda, Bryan Mesias, Roxana Serrano, Geidy Alessi, Dario R. Beach, Thomas G. Benson, Deanna L. White, Charles L. Cowell, Rita M. Das, Sonal S. West, Andrew B. Melrose, Heather L. Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title | Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title_full | Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title_fullStr | Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title_full_unstemmed | Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title_short | Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies |
title_sort | comprehensive characterization and optimization of anti-lrrk2 (leucine-rich repeat kinase 2) monoclonal antibodies |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682752/ https://www.ncbi.nlm.nih.gov/pubmed/23560750 http://dx.doi.org/10.1042/BJ20121742 |
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