Fast and easy single-molecule pulldown assay based on agarose microbeads

Abstract

SUMMARYThe recently developed single-molecule pulldown (SiMPull) assay by Jain and colleagues is a highly innovative technique but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-noise ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of powerful and versatile SiMPull in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used this method in the study of rare auditory hair cells for the first time.

Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding

“…Our microbead-based SiMPull technique (12) was instrumental in the in vivo analysis of the expression and interaction of TMC1 and LHFPL5 in this study. Several components of the MT complex in hair cells have been identified in the last 10 y, and we can reasonably expect additional components to be identified in future studies.…”

“…Because of the scarceness of the raw material (hair cells), previous biochemical studies of MT-complex components have relied mainly on heterologous overexpression systems; however, these systems are prone to artifacts, and the obtained results carry little weight (in most research fields) in the absence of validation through studies of endogenous proteins. The microbead-based SiMPull method (12) substantially simplifies the original SiMPull assay invented by Jain et al (36) and makes the application of the powerful SiMPull technique in regular laboratories considerably more feasible. Notably, SiMPull potentially can also be used for studying the stoichiometry and the interaction kinetics of MT-complex proteins (12).…”

“…The microbead-based SiMPull method (12) substantially simplifies the original SiMPull assay invented by Jain et al (36) and makes the application of the powerful SiMPull technique in regular laboratories considerably more feasible. Notably, SiMPull potentially can also be used for studying the stoichiometry and the interaction kinetics of MT-complex proteins (12). Our hope is that this technique will markedly facilitate the biochemical investigation of MT-complex proteins and other critical nonabundant proteins in hair cells.…”

“…Microbead-Based SiMPull. Detailed experimental principles and procedures for SiMPull are described in the study by Zhao et al (12).…”

“…Here, we investigate the physical and functional interactions between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay (12). We demonstrate that TMC1 physically interacts with LHFPL5 and that TMC1 expression is stabilized by LHFPL5 binding both in heterologous expression systems and in hair cells.…”