There is a need for standardized validation methods for antibody specificity and selectivity. In this paper, the researchers show that the five pillars involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies and capture mass spectrometry analysis, can be used to validate antibodies for Western blot applications in a standardized and systematic manner. The study was led by Mathis Uhlén, KTH/SciLifeLab.
“One of the main focuses in this work has been to introduce orthogonal methods for validation of antibodies.” Said Fredrik Edfors, currently at Stanford University, first author of the study. “This strategy opens up streamlined efforts to validate antibodies using verified and standardized cell- or tissue lysate panels centrally obtained by certified providers, in which the antibody-independent method is performed and the data used for validation is provided and shared in an open access manner.”
The results of the study, published in Nature Communications, show that more than 6,000 antibodies published in the Human Protein Atlas could be validated in this using at least one of the five strategies presented. More than 1,600 of the antibodies were validated by at least two of the pillars and 267 with three or more pillars.
The researchers also show that all the validation strategies described in the paper can be used to investigate and validate antibodies that yield several bands on the Western blot assay.
“This phenomenon is not uncommon and could either be due to unspecific staining as a result of off-target binding or specific staining of multiple isoforms of the target protein due to proteolysis, post-translational modifications or splice variants. This includes identification and label-free quantification of proteins and their migration in the gel for 5,605 human target proteins.” Said Fredrik Edfors.
Read full scientific paper in Nature Communications