Bradner James E, McPherson Olivia M, Koehler Angela N
Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Nat Protoc. 2006;1(5):2344-52. doi: 10.1038/nprot.2006.282.
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.
本方案描述了一种以微阵列形式共价捕获具有不同反应性功能基团的小分子的可靠方法，并概述了用感兴趣的蛋白质探测小分子微阵列（SMM）的程序。采用蒸汽催化、异氰酸酯介导的表面固定方案来连接生物活性小分子、天然产物以及源自多样性导向合成途径的小分子。此外，还描述了一种用纯化蛋白质和细胞裂解物筛选SMM的优化方法。最后，提供了一种与商用软件兼容的数据分析建议模型。这些程序实现了一个平台功能，可用于发现新的相互作用，潜在应用于免疫球蛋白谱分析、细胞状态的比较分析和配体发现。使用合适的材料和实验设置，SMM的打印可在3天内14小时完成。筛选和数据分析需要2天。提供了详细的时间表。
