RT Journal Article
SR Electronic
T1 Fast and easy single-molecule pulldown assay based on agarose microbeads
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.09.20.305177
DO 10.1101/2020.09.20.305177
A1 Qirui Zhao
A1 Yusheng Shen
A1 Xiaofen Li
A1 Fang Tian
A1 Xiaojie Yu
A1 Levent Yobas
A1 Hyokeun Park
A1 Pingbo Huang
YR 2020
UL http://biorxiv.org/content/early/2020/09/20/2020.09.20.305177.abstract
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.