A proposal for validation of antibodies

Abstract

We convened an ad hoc International Working Group for Antibody Validation in order to formulate the best approaches for validating antibodies used in common research applications and to provide guidelines that ensure antibody reproducibility. We recommend five conceptual ‘pillars’ for antibody validation to be used in an application-specific manner.

Figure 1 |

Validation of antibodies in western blot applications using genetic strategies (a), orthogonal strategies (b), independent antibody strategies (c) and tagged protein expression (d). (a) The western blot against human C9orf78 compares siRNA knockdown of protein in cell line (labeled 1 and 2) with control (without siRNA, labeled C). Percent staining of the band is indicated. M, size marker, (b) Simultaneous analysis of human STOM abundance in eight cell lines (labeled 1–8) using western blot (WB) and targeted proteomics (MS, mass spectrometry) resulting in a Pearson correlation of 0.97 (P = 0.00006). (c) Simultaneous western blot analysis of human PRKCA in eight cell lines using two independent antibodies (Ab1 and Ab2, 1 and 2) resulting in a Pearson correlation of 0.93 (P = 0.0009). (d) A fusion of human IL-8 with a peptide tag was stained with either the antibody to be validated (Ab) or a tag-specific antibody (Tag) in cell line samples (labeled 1–4). The black arrows indicate the theoretical sizes of the target protein.