The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence

Riham Ayoubi; Walaa Alshafie; Irina Shlaifer; Kathleen Southern; Peter S McPherson; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group

doi:10.12688/f1000research.132628.2

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence

F1000Res. 2024 Jul 10:12:324. doi: 10.12688/f1000research.132628.2. eCollection 2023.

Authors

Riham Ayoubi¹, Walaa Alshafie¹, Irina Shlaifer², Kathleen Southern¹, Peter S McPherson¹, Carl Laflamme¹; NeuroSGC/YCharOS/EDDU collaborative group

Affiliations

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada.
² The Neuro's Early Drug Discovery Unit (EDDU), Structural Genomics Consortium, McGill University, Montreal, Québec, H3A 2B4, Canada.

Abstract

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Keywords: SQSTM1; Sequestosome-1; Uniprot ID Q13501; Western blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation.

MeSH terms

Antibodies / immunology
Blotting, Western*
Fluorescent Antibody Technique* / methods
Humans
Immunoprecipitation* / methods
Sequestosome-1 Protein* / immunology
Sequestosome-1 Protein* / metabolism

Substances

Sequestosome-1 Protein
Antibodies
SQSTM1 protein, human

Grants and funding

This work was supported in part by the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP is a private-public partnership created by the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. The grant was from a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda. RA and WA were supported by a Mitacs fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.