Quantification of Fluorescence Signals in Purified Mitochondria

Abstract

Studies of localization and transient association of proteins with mitochondria are limited by the lack of appropriate purification, high resolution imaging and quantification techniques. Here, we investigated the localization of mitochondrial proteins and quantified them in isolated mitochondria from murine heart. Mitochondria were rapidly isolated and purified using a Percoll gradient. The degree of purity of three fractions (M1-M3) was verified by Western blots using markers of different cellular components along with mitochondrial markers. M3 fraction was the purest with negligible amounts of contaminating proteins from the plasma membrane (0.2%), nucleus (6.5%), Golgi complex (1.8%), and endoplasmic reticulum (2.2%) as compared to the whole cell lysate fractions, but was enriched with mitochondrial markers VDAC1 (126%) and COX4 (139%). Thus, M3 fraction was used for sequential labeling with Mitotracker (100 µM) and specific antibodies for mitochondrial proteins. Images were obtained using confocal microscopy. To extract signal information of distinct mitochondria, we utilized the Statistical Region Merging (SRM) and Robust Automatic Threshold Selection (RATS) algorithms to minimize the human bias introduced by the commonly used empiric thresholding. The criteria to positively identify isolated mitochondria were: Mitotracker labeling, average intensity and size. Once mitochondria were selected, the degree of protein co-labeling was quantified. As a proof of concept, we show that VDAC1 and COX4 highly colocalize with Mitotracker labeled mitochondria but not alpha 1C Ca channel. These techniques pave the way to further study mitochondrial proteins and their temporal association with non-mitochondria proteins. Supported by NIH.