Abstract
Lysate microarrays (reverse-phase protein arrays) hold great promise as a tool for systems-level investigations of signaling and multiplexed analyses of disease biomarkers. To date, however, widespread use of this technology has been limited by questions concerning data quality and the specificity of detection reagents. To address these concerns, we developed a strategy to identify high-quality reagents for use with lysate microarrays. In total, we tested 383 antibodies for their ability to quantify changes in protein abundance or modification in 20 biological contexts across 17 cell lines. Antibodies yielding significant differences in signal were further evaluated by immunoblotting and 82 passed our rigorous criteria. The large-scale data set from our screen revealed that cell fate decisions are encoded not just by the identities of proteins that are activated, but by differences in their signaling dynamics as well. Overall, our list of validated antibodies and associated protocols establish lysate microarrays as a robust tool for systems biology.
Highlights
From the ‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, Massachusetts 02138, §Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115
In our own work [13], which focused on a single cell line, we started with a set of 61 commercial antibodies and found that only 12 of them yielded data on lysate microarrays that matched those collected by quantitative Western blotting
Design of a High-throughput Screen for Functional Detection Antibodies—An antibody can be used with lysate microarrays if it meets two criteria: [1] it produces a significant difference in signal across the samples of interest, and [2] these differences correspond to changes in the levels of the target antigen
Summary
Lysate Microarrays Enable High-throughput, Quantitative Investigations of Cellular Signaling*□S. Thousands of biological specimens can be arrayed onto hundreds of membrane-coated slides, each of which can be queried with a different detection antibody This format allows dense sampling of information at a protein level and in a high-throughput fashion. Several groups have used this technology to study biological systems [12, 14] and standardized protocols have been published [15], lysate microarrays have not yet gained wide-spread adoption, largely owing to questions regarding data quality and the limited availability of highly validated detection antibodies. If the two data sets agree, the antibody is considered validated for use with that cellular background Using this strategy, we screened 383 commercial antibodies and successfully validated 82 of them in one or more biological context. This study focused on characterizing antibodies for lysate microarrays, our overall strategy is general and can be applied to other high-throughput immunoaffinity assays as well
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