A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence

F1000Res. 2023 Dec 28:12:1578. doi: 10.12688/f1000research.143928.2. eCollection 2023.

Abstract

Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson's disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords: RAB1A and RAB1B; Rab1A and Rab1B; Uniprot ID P62820 (Rab1A) and Q9H0U4 (Rab1B); Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation.

MeSH terms

  • Blotting, Western
  • Fluorescent Antibody Technique
  • Humans
  • Immunoprecipitation
  • Proteins*
  • Reproducibility of Results
  • rab1 GTP-Binding Proteins*

Substances

  • Proteins
  • Rab1B protein, human
  • rab1 GTP-Binding Proteins

Grants and funding

The grant was from a Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). RA is 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.