Validation of antibodies for Western blot applications using orthogonal methods
(English)Manuscript (preprint) (Other academic)
There is a great need for standardized validation methods for antibody specificity and selectivity. Here, we describe the use of orthogonal methods in which the specificity of an antibody in a particular application is determined based on correlation of protein abundance across several samples using an antibody-independent method. We show that pair-wise correlation between orthogonal samples can be used to score the specificity of antibodies in a standardized manner using a test panel of human cell lines. Here, we investigated two independent methods for validation of antibodies in Western blot applications, namely transcriptomics and targeted proteomics and we show that the two methods yield similar, but not identical results. The orthogonal methods can also be used to investigate on- and off- target binding for antibodies with multiple bands in the Western blot assay. In conclusion, orthogonal methods for antibody validation provide an attractive strategy for systematic validation of antibodies in a quantitative manner.
Biochemistry and Molecular Biology
Research subject Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-193962OAI: oai:DiVA.org:kth-193962DiVA: diva2:1034916
QC 201610132016-10-132016-10-132016-10-13Bibliographically approved