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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)
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
Keywords
Protein Gabarap; Autophagy; Receptor; Expression; Family; Atg8; Bafilomycins; Trafficking; Sensors; Lc3
Language
english
Publication Year
2019
HGF-reported in Year
2019
ISSN (print) / ISBN
2045-2322
e-ISSN
2045-2322
Journal
Scientific Reports
Quellenangaben
Volume: 9,
Issue: 1,
Article Number: 526,
Supplement: https://doi.org/10.1038/s41598-018-36717-1.
Publisher
Nature Publishing Group
Publishing Place
London
Reviewing status
Peer reviewed
Institute(s)
CF Monoclonal Antibodies (CF-MAB)
Institute of Molecular Immunology (IMI)
Institute of Molecular Immunology (IMI)
POF-Topic(s)
30201 - Metabolic Health
30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
Research field(s)
Helmholtz Diabetes Center
Immune Response and Infection
Immune Response and Infection
PSP Element(s)
G-502210-001
G-501793-001
G-501793-001
WOS ID
WOS:000456553400091
Scopus ID
85060531005
PubMed ID
30679523
Erfassungsdatum
2019-03-07