PT  - JOURNAL ARTICLE
AU  - Qirui Zhao
AU  - Yusheng Shen
AU  - Xiaofen Li
AU  - Fang Tian
AU  - Xiaojie Yu
AU  - Levent Yobas
AU  - Hyokeun Park
AU  - Pingbo Huang
TI  - Fast and easy single-molecule pulldown assay based on agarose microbeads
AID  - 10.1101/2020.09.20.305177
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.09.20.305177
4099  - http://biorxiv.org/content/early/2020/09/20/2020.09.20.305177.short
4100  - http://biorxiv.org/content/early/2020/09/20/2020.09.20.305177.full
AB  - The 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.Competing Interest StatementThe authors have declared no competing interest.