Abstract
This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.
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Acknowledgements
The project has been funded in whole or in part with Federal funds from the National Cancer Institute's Initiative for Chemical Genetics, National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Service, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. We thank R. Mazitschek for donation of AP1497 derivatives. J.E.B. is supported by the Multiple Myeloma Research Foundation and the Clinical Investigator Training Program: Harvard–MIT Health Sciences and Technology–Beth Israel Deaconess Medical Center, in collaboration with Pfizer and Merck & Company.
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Supplementary information
Supplementary Video 1
Parafilm Screening Methods. (AVI 3475 kb)
Supplementary Video 2
Small Molecule Microarrays (MOV 5969 kb)
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Bradner, J., McPherson, O. & Koehler, A. A method for the covalent capture and screening of diverse small molecules in a microarray format. Nat Protoc 1, 2344–2352 (2006). https://doi.org/10.1038/nprot.2006.282
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DOI: https://doi.org/10.1038/nprot.2006.282
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