Enhanced validation of antibodies for research applications.

Edfors F, Hober A, Linderbäck K, Maddalo G, Azimi A, Sivertsson Å, Tegel H, Hober S, Szigyarto CA, Fagerberg L, von Feilitzen K, Oksvold P, Lindskog C, Forsström B, Uhlen M

Nat Commun 9 (1) 4130 [2018-10-08; online 2018-10-08]

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

Bioinformatics Compute and Storage [Service]

NGI Stockholm (Genomics Applications) [Service]

NGI Stockholm (Genomics Production) [Service]

National Genomics Infrastructure [Service]

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PubMed 30297845

DOI 10.1038/s41467-018-06642-y

Crossref 10.1038/s41467-018-06642-y


pmc PMC6175901

BioProject PRJNA183192 [RNA sequencing data for human cell lines.]

BioProject https://www.ncbi.nlm.nih.gov/bioproject/PRJNA183192 [RNA sequencing data for human cell lines.]

https://panoramaweb.org/western_blot.url [Enhanced validation of antibodies for research applications(Chromatograms)]

PXD010178 [Enhanced validation of antibodies for research applications(Capture MS LC-MS/MS data)]

PXD005914 [The goal is to define guidelines for Ab western blot validation (TMT10plex data)]