Quantification of Protein Copy Number in Single Mitochondria

Bcl-2 family proteins, represented by the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax, are key regulators of the mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome C release, it is essential to know the number of proteins in individual mitochondria. Employing an immunofluorescent labelling strategy, the copy number and distribution of proteins in single mitochondria are accessed via the Flow NanoAnalyzer.

Figure 1. Quantitative measurement of Bcl-2 copy number in individual mitochondria.

A series of fluorescent nanospheres with fluorescence intensity calibrated by the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve. The fluorescence intensity of a single mitochondrion could then be converted to the number of proteins per mitochondrion.

Biosens. Bioelectron., 2015, 74, 476-482.