pPAmCherry-Mito Vector is a mammalian expression vector encoding PAmCherry, a photoactivatable mutant of the fluorescent protein mCherry, fused to the mitochondrial targeting sequence derived from the precursor subunit VIII of human cytochrome C oxidase. PAmCherry is non-fluorescence until photoactivated by a short exposure to light at a wavelength between 350 nm and 400 nm. The excitation/emission wavelengths of photoactivated PAmCherry are 564 nm and 595 nm.
Photoactivated PAmCherry-Mito shows strong red fluorescence and localizes correctly to the mitochondria. U2OS cells were transiently transfected with pPAmCherry-Mito. PAmCherry-Mito was activated using an Argon 458 nm laser (scan speed: 400 Hz), and the cells were imaged using a HeNe 543 nm laser for excitation and a standard red fluorescence emission filter set.
The mCherry monoclonal antibody detects PAmCherry-N1 in the lysate of mammalian cells. HEK 293 cells were transiently transfected with pPAmCherry-N1. Cell lysates (corresponding to 30,000 cells) were prepared from HEK 293 cells transiently expressing PAmCherry-N1 (Lane 1) or a negative control (untransfected cells; Lane 2). Both lysates and a positive control (5 ng recombinant mCherry; Lane 3) were separated by SDS-PAGE and analyzed by Western blot using the mCherry monoclonal antibody at the recommended dilution of 1:1000.
Prior to photoactivation, no red fluorescence is detected in a cell expressing PAmCherry-Mito (Panel A). However, after photoactivation, strong red fluorescence is observed in the subcellular, activated region of the cell (Panel B). U2OS cells were transiently transfected with pPAmCherry-Mito. Cells were imaged prior to activation in order to determine the level of background fluorescence (Panel A). PAmCherry-Mito was then activated in a small region of a cell imaged using a HeNe 543 nm laser for excitation and a standard red fluorescence emission filter set (Panel B).
PAmCherry-Mito makes it easy to follow the behavior of a subset of mitochondria. Activating the mitochondria in just one region of the cell makes it possible to follow their movements into dark (nonactivated) areas of the cell. U2OS cells were transiently transfected with pPAmCherry-Mito. PAmCherry-Mito was activated in a small region of a cell, and the cells were imaged every 10 sec for 15 min using a HeNe 543 nm laser for excitation and a standard red fluorescence emission filter set.
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