THE HUMAN PROTEIN ATLAS BLOG
Validation of antibodies for bioimaging
In a recent publication in Journal of Proteome Research, researchers from the Cell Profiling group of the Human Protein Atlas team present a new approach for validation of antibodies for bioimaging applications.
Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here the researchers present a strategy to systematically validate antibodies for immunofluorescence applications using gene tagging.
Marie Skogs is the first author of the study.
– After a M. Sc. in Biotechnological Engineering from KTH – The Royal Institute of Technology I joined the Human Protein Atlas Project as a research engineer working with the Cell Atlas. After a few years I started my PhD studies as a part of the Cell Atlas Project as well.
The project described here was a team effort by several members of the Cell Atlas team, where Marie Skogs was the main responsible for planning the lab work and writing the manuscript.
– We designed a systematic scheme for assessing antibody on- and off-target binding in immunofluorescence applications and used it to investigate the performance of 197 HPA antibodies, Marie Skogs explains.
The antibody performance in immunofluorescence was compared with the performance in Western Blot. Finally, polyclonal batch-to-batch variations were assessed for 35 antibodies, where a second batch of antibody was produced by immunization of the same antigen in another rabbit.
– We conclude that expression of a tagged protein at endogenous levels is an attractive path for systematic identification and validation of antibodies in bioimaging applications.
Since antibody performance differs across applications and contexts the researchers stress the fact that antibodies should be validated using the same application and context as its intended use and we show that detection of GFP-tagged proteins is a good approach for systematic validations.
– The validation strategy presented in this article is one of the strategies used to validate the data presented in the Cell Atlas, and a valuable complement to silencing/knockdown in order to cover all genes, Marie Skogs concludes.
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Frida Henningson Johnson