High-Throughput Single-Cell Analysis of Low Copy Number Protein

Single-cell analysis is vital in providing insights into the heterogeneity of molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. β-galactosidase (β-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here, the Flow NanoAnalyzer is used for the development of a high-throughput method for the single-cell analysis of low copy number β-gal proteins. Upon fluorescence staining with a fluorogenic substrate C12FDG, quantitative measurements of the basal and near-basal expression of β-gal in single bacteria are demonstrated. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration, the β-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell.

Built upon the sensitivity and speed of the instrument and the good cell retention of the hydrolysis, products of C12FDG, β-gal are detected at single bacterial cell level.

Biosens. Bioelectron., 2013, 48, 49-55.