Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells

Autophagy. 2023 Sep;19(9):2575-2577. doi: 10.1080/15548627.2023.2213515. Epub 2023 May 16.

Abstract

LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.

Keywords: Antibody; CRISPR-Cas9; LAMP2A; cancer; chaperone-mediated autophagy; immunofluorescence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies
  • Autophagy
  • Chaperone-Mediated Autophagy*
  • Humans
  • Lysosomal-Associated Membrane Protein 2 / genetics
  • Lysosomal-Associated Membrane Protein 2 / metabolism
  • Lysosomes / metabolism
  • Neoplasms* / genetics
  • Neoplasms* / metabolism

Substances

  • Lysosomal-Associated Membrane Protein 2
  • Antibodies

Grants and funding

This work was funded by grants from Karolinska Institutet, the Swedish Research Council (VR 2019-01535), and the Swedish Cancer Society (20 1016 Pj). We especially thank Abcam for their collaboration, technical support, and advice.