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      A proposal for validation of antibodies

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          Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.

          Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
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            Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

            Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.
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              An open letter to our readers on the use of antibodies.

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                Author and article information

                Journal
                Nature Methods
                Nat Methods
                Springer Science and Business Media LLC
                1548-7091
                1548-7105
                October 2016
                September 5 2016
                October 2016
                : 13
                : 10
                : 823-827
                Article
                10.1038/nmeth.3995
                27595404
                625d2fde-58d6-4a64-8df6-7c4828cea8a9
                © 2016

                http://www.springer.com/tdm

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