Protocol for systematic identification of microtubule luminal proteins using knock-in BioID proximity labeling and fluorescence imaging validation.

Systematic elucidation of microtubule (MT) luminal proteins provides insights into MT biology and its molecular regulation. Here, we present a protocol for screening and validating these proteins using proximity labeling, mass spectrometry, fluorescence imaging and western blot (WB). We describe steps for constructing a biotin ligase knock-in cell line and performing BioID screening. We detail how to perform fluorescence imaging and WB to validate the MT luminal proteins. This protocol enables systematic identification to advance research on MT luminal proteins. For complete details on the use and execution of this protocol, please refer to Shao et al.1.