A high-throughput pipeline for validation of antibodies

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).This paper describes a platform for carrying out antibody-based capture and mass spectrometry in parallel, and tests the feasibility of this platform for high-throughput validation of antibodies.

[1]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[2]  S. Shen-Orr,et al.  Social network architecture of human immune cells unveiled by quantitative proteomics , 2017, Nature Immunology.

[3]  Andreas Plückthun,et al.  Reproducibility: Standardize antibodies used in research , 2015, Nature.

[4]  Monya Baker,et al.  Antibody anarchy: A call to order , 2015, Nature.

[5]  E. Lundberg,et al.  A Genecentric Human Protein Atlas for Expression Profiles Based on Antibodies* , 2008, Molecular & Cellular Proteomics.

[6]  Tomáš Kalina,et al.  An automated analysis of highly complex flow cytometry‐based proteomic data , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[7]  M. Mann,et al.  Comparative Proteomic Analysis of Eleven Common Cell Lines Reveals Ubiquitous but Varying Expression of Most Proteins* , 2012, Molecular & Cellular Proteomics.

[8]  David Maier,et al.  A call to order , 1993, PODS '93.

[9]  Jeffrey R. Whiteaker,et al.  Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation , 2015, Nature Methods.

[10]  J. Harkins,et al.  Using the GELFREE 8100 Fractionation System for molecular weight-based fractionation with liquid phase recovery. , 2009, Journal of visualized experiments : JoVE.

[11]  Tomas Kalina,et al.  Multiplexed immuno‐precipitation with 1725 commercially available antibodies to cellular proteins , 2011, Proteomics.

[12]  E. Lundberg,et al.  Antibody performance in western blot applications is context-dependent. , 2014, Biotechnology Journal.

[13]  Fridtjof Lund-Johansen,et al.  Antibody Array Analysis with Label-based Detection and Resolution of Protein Size *S , 2009, Molecular & Cellular Proteomics.

[14]  M. Baker.  Reproducibility crisis: Blame it on the antibodies , 2015, Nature.

[15]  B. Kuster,et al.  A bead-based western for high-throughput cellular signal transduction analyses , 2016, Nature Communications.

[16]  Mathias Wilhelm,et al.  Global proteome analysis of the NCI-60 cell line panel. , 2013, Cell reports.

[17]  F. Lund-Johansen,et al.  Should we ignore western blots when selecting antibodies for other applications? , 2017, Nature Methods.

[18]  Amber L. Couzens,et al.  The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data , 2013, Nature Methods.

[19]  Emma Lundberg,et al.  A proposal for validation of antibodies , 2016, Nature Methods.

[20]  M. Mann.  Can proteomics retire the western blot? , 2008, Journal of proteome research.