title: Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.
abstract: Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Marie
authors.initials: M
authors.lastname: Skogs
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Stadler
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Rutger
authors.initials: R
authors.lastname: Schutten
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Hjelmare
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Christian
authors.initials: C
authors.lastname: Gnann
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Björk
authors.affiliation: Molecular Cell Biology and Genetics, Max Planck Institute , D- 01307 Dresden, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Poser
authors.affiliation: Molecular Cell Biology and Genetics, Max Planck Institute , D- 01307 Dresden, Germany.
authors.forename: Anthony
authors.initials: A
authors.lastname: Hyman
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.affiliation: Science for Life Laboratory, Royal Institute of Technology , SE-114 28 Stockholm, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Lundberg
mesh_terms: D000704:Analysis of Variance; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry; D001271:Atlases as Topic; D002460:Cell Line; D005455:Fluorescent Antibody Technique / Q000592:standards*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000906:Antibodies; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Cell Atlas; GFP; Human Protein Atlas; antibody validation; confocal microscopy; immunofluorescence; spatial proteomics; subcellular localization