Single-cell western blotting.

ABSTRACT: To measure cell-to-cell variation in protein-mediated functions, we developed an approach to conduct ?10(3) concurrent single-cell western blots (scWesterns) in ?4 h. A microscope slide supporting a 30-?m-thick photoactive polyacrylamide gel enables western blotting: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins and antibody probing. We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation. The scWestern quantified target proteins even with off-target antibody binding, multiplexed to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supported analyses of low starting cell numbers (?200) when integrated with FACS. The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

SUBMITTER: Hughes AJ 

PROVIDER: S-EPMC4077215 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature