Identification of the protein targets of bioactive small molecules is a routine challenge in chemical biology and phenotype-based drug discovery. Recent years have seen an explosion of approaches to meeting this challenge, but the traditional method of affinity pulldowns remains a practical choice in many contexts. This technique can be used as long as an affinity probe can be synthesized, usually with a crosslinking moiety to enable photo-affinity pulldowns. It can be applied to varied tissue types and can be performed with minimal specialized equipment. Here, we provide our protocol for photo-affinity pulldown experiments, with notes on making this method generally applicable to varied target identification challenges.
Keywords: Biotin; Click chemistry; Inhibitor; Mass spectrometry; Natural product; Off-target; Photoreactive group; Small molecule; Synthetic chemistry; Target identification.
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