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

Authors :
Hyokeun Park
Pingbo Huang
Qirui Zhao
Fang Tian
Yusheng Shen
Xiaofen Li
Levent Yobas
Xiaojie Yu
Publication Year :
2020
Publisher :
Cold Spring Harbor Laboratory, 2020.

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.

Details

Database :
OpenAIRE
Accession number :
edsair.doi...........42af1bfaf017f6dc2cda69553bc5280b