Peroxisome structures are single membrane-bound vesicles that are involved in energy metabolism and lipid biosynthesis. Here we describe peroxisome stains using organelle-specific fluorescent fusion proteins in live cells or antibodies to peroxisome membrane proteins in fixed cells.
Peroxisomes are single membrane–bound vesicles found in most eukaryotic cells. They contain several different enzymes and are involved in various metabolic pathways . Most importantly, peroxisomes are involved in the oxidation of fatty acids, a major source of metabolic energy. Additionally, peroxisomes catalyze the breakdown of reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2), by converting it to water, or using it to oxidize other organic compounds [1,2]. Peroxisomes are also critical in the biosynthesis of phospholipids involved in maintaining the normal function of both brain and lungs in mammals.
Expression of fluorescent fusion protein
|Secondary staining of peroxisomes|
Uses peroxisomal C-terminal targeting sequence
|Labels peroxisomal membrane protein 70 (PMP70)|
|Common filter set|
Alexa Fluor 488
CellLight fluorescent fusion proteins can be used as peroxisome labels in live cells and to follow the dynamics of intracellular behavior. CellLight Peroxisome proteins are ready-to-use constructs that use the human peroxisomal C-terminal targeting sequence (0.3 kDa) to target the peroxisome.
Introducing CellLight fluorescent proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These peroxisome fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells (Figures 1 and 2), or after formaldehyde fixation for colocalization studies.
The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit provides the reagents needed to label peroxisomes in fixed cells, including cell fixative and permeabilization reagents (Figure 3). For detection, the kit employs a primary antibody directed against peroxisomal membrane protein 70 (PMP70), which is a high-abundance, integral-membrane component of peroxisomes. PMP70 is significantly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid beta-oxidation enzymes.
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