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Simons, I.M.* ; Mohrlüder, J.* ; Feederle, R. ; Kremmer, E. ; Zobel, T.* ; Dobner, J.* ; Bleffert, N.* ; Hoffmann, S.* ; Willbold, D.*

The highly GABARAP specific rat monoclonal antibody 8H5 visualizes GABARAP in immunofluorescence imaging at endogenous levels.

Sci. Rep. 9, https://doi.org/10.1038/s41598-018-36717-1.:526 (2019)
Publ. Version/Full Text Research data DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Protein Gabarap; Autophagy; Receptor; Expression; Family; Atg8; Bafilomycins; Trafficking; Sensors; Lc3
ISSN (print) / ISBN 2045-2322
e-ISSN 2045-2322
Journal Scientific Reports
Quellenangaben Volume: 9, Issue: 1, Pages: , Article Number: 526, Supplement: https://doi.org/10.1038/s41598-018-36717-1.
Publisher Nature Publishing Group
Publishing Place London
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) CF Monoclonal Antibodies (CF-MAB)
Institute of Molecular Immunology (IMI)