Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins

QC 20170216
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.

Stadler, CharlotteKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1r100ik (författare)
Schutten, RutgerKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1sr8wj5 (författare)
Hjelmare, MartinKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1jdseac (författare)
Gnann, ChristianKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1338pp0 (författare)
Björk, LarsKarolinska Institutet,KTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1ip3p7j (författare)
Poser, Ina (författare)
Hyman, Anthony (författare)
Uhlén, MathiasKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u1dulvmw (författare)
Lundberg, EmmaKTH,Science for Life Laboratory, SciLifeLab(Swepub:kth)u12bylj1 (författare)
KTHScience for Life Laboratory, SciLifeLab (creator_code:org_t)

Ingår i:Journal of Proteome Research: American Chemical Society (ACS)16:1, s. 147-1551535-38931535-3907

Av författaren/redakt...: Skogs, Marie; Stadler, Charlot ...; Schutten, Rutger; Hjelmare, Martin; Gnann, Christian; Björk, Lars; visa fler...; Poser, Ina; Hyman, Anthony; Uhlén, Mathias; Lundberg, Emma; visa färre...

Om ämnet

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.