Assay reproducibility demonstrated by experiments conducted on three separate days. HepG2 cells were grown for 7 days in the presence of 10 µM chloramphenicol and then measured in duplicate on 3 different days using the MitoBiogenesis™ In-Cell ELISA Kit. The assay was able to record inhibition of mitochondrial biogenesis with an average intra-day CV of 2.3% and an average day-to-day CV of 1.9%.

Visual representation of inhibition of mtDNA-encoded protein synthesis. Visually-imaged levels of mtDNA-encoded COX-I are shown in the green 800 channel while levels of nuclear DNA-encoded SDH-A are shown in the red 700 channel. The merged image shows the relative ratios of COX-I/SDH-A protein expression in each microwell. Wells with normal levels of both proteins exhibit a yellow merged color due to approximately equal red and green signals. In contrast, wells with low levels of COX-I and normal levels of SDH-A exhibit an orange color due to the relative lack of green (COX-I) fluorescence. The specific inhibition of mtDNA-encoded protein synthesis by chloramphenicol is thus easily observed.

Quantitative measurement of the COX-I/SDH-A protein expression ratio. At all cell concentrations, a constant ratio of mtDNA-encoded protein expression (COX-I) to nuclear DNA-encoded mitochondrial protein expression (SDH-A) is observed in untreated cells. Therefore, normalizing COX-I levels to SDH-A levels simplifies data analysis and eliminates the need to perform all tests at the same cell concentration.

Antibody specificity demonstrated by immunocytochemistry. Two-color immunocytochemical labeling of cultured cells with the two MS642 primary monoclonal antibodies specific for COX-I and SDH-A. The two antibodies exhibit striking and specific co-localization in the mitochondria, consistent with the known mitochondrial expression of both proteins.

Antibody specificity demonstrated by Western Blot. A Western blot of total cell protein (10 µg) from human or rat cultured cells was probed with the primary and secondary antibodies and scanned with a LI-COR® Odyssey® imager. The two mitochondrial proteins targeted by the two primary mAbs were labeled and visualized specifically despite the presence of thousands of other proteins. Furthermore, reduction of mtDNA levels in human Rho0 (mtDNA-depleted) cells, or inhibition of mitochondrial protein translation by chloramphenicol in rat cells both result in specific reduction of COX-I protein while nuclear DNA-encoded SDH-A is unaffected.

Cells are grown to ~80% confluency in a 96- or 384-well plate, a drug/other treatment is applied to stimulate a cellular response. The cells are then fixed and permeabilized, effectively "freezing" them. Primary antibodies are then added which bind to their intended targets within the mitochondria or other subcellular compartment. After incubation, the unbound primary antibodies are washed away and secondary antibodies are added. These secondaries are conjugated to either IRDyes® or to an enzyme label (HRP or AP) for the colorimetric versions of the assays. Unbound secondaries are washed away, reaction buffer is added for the colorimetric assays, and the signal is read on a suitable instrument for the kit type.» In-cell ELISA diagram in PDF format