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Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.

Publication ,  Journal Article
Davies, P; Hinkle, KM; Sukar, NN; Sepulveda, B; Mesias, R; Serrano, G; Alessi, DR; Beach, TG; Benson, DL; White, CL; Cowell, RM; Das, SS ...
Published in: Biochem J
July 1, 2013

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.

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Published In

Biochem J

DOI

EISSN

1470-8728

Publication Date

July 1, 2013

Volume

453

Issue

1

Start / End Page

101 / 113

Location

England

Related Subject Headings

  • Rats
  • Rabbits
  • Protein Serine-Threonine Kinases
  • Parkinson Disease
  • Mice, Knockout
  • Mice
  • Male
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • Immunoblotting
  • Humans
 

Citation

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Davies, P., Hinkle, K. M., Sukar, N. N., Sepulveda, B., Mesias, R., Serrano, G., … Melrose, H. L. (2013). Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies. Biochem J, 453(1), 101–113. https://doi.org/10.1042/BJ20121742
Davies, Paul, Kelly M. Hinkle, Nour N. Sukar, Bryan Sepulveda, Roxana Mesias, Geidy Serrano, Dario R. Alessi, et al. “Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.Biochem J 453, no. 1 (July 1, 2013): 101–13. https://doi.org/10.1042/BJ20121742.
Davies P, Hinkle KM, Sukar NN, Sepulveda B, Mesias R, Serrano G, et al. Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies. Biochem J. 2013 Jul 1;453(1):101–13.
Davies, Paul, et al. “Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.Biochem J, vol. 453, no. 1, July 2013, pp. 101–13. Pubmed, doi:10.1042/BJ20121742.
Davies P, Hinkle KM, Sukar NN, Sepulveda B, Mesias R, Serrano G, Alessi DR, Beach TG, Benson DL, White CL, Cowell RM, Das SS, West AB, Melrose HL. Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies. Biochem J. 2013 Jul 1;453(1):101–113.

Published In

Biochem J

DOI

EISSN

1470-8728

Publication Date

July 1, 2013

Volume

453

Issue

1

Start / End Page

101 / 113

Location

England

Related Subject Headings

  • Rats
  • Rabbits
  • Protein Serine-Threonine Kinases
  • Parkinson Disease
  • Mice, Knockout
  • Mice
  • Male
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • Immunoblotting
  • Humans