Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies.
Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94% of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ∼2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.
Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA.
Thane H
TH
Taylor
Nicholas W
NW
Frost
Michael T
MT
Bowser
Edgar A
EA
Arriaga
D000906:Antibodies / Q000032:analysis*; D045744:Cell Line, Tumor; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008164:Luminescent Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry / Q000648:ultrastructure*; D033681:Mitochondrial Membrane Transport Proteins / Q000032:analysis*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C489971:TOMM22 protein, human; C413663:fluorescent protein 583