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Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation‐mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase‐catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K‐BILDS). As described in these protocols, K‐BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ‐phosphate‐modified ATP analog ATP‐biotin for kinase‐catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K‐BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K‐BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. 

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase‐catalyzed crosslinking and immunoprecipitation (K‐CLIP). K‐CLIP uses a photocrosslinking γ‐phosphoryl‐modified ATP analog, such as ATP‐arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase‐substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K‐CLIP is an adaptable chemical tool to investigate and discover kinase‐substrate pairs, which will promote characterization of complex phosphorylation‐mediated cell biology.