The Premo™ Autophagy Sensor RFP-p62 Kit combines the ability to monitor the induction or inhibition of autophagy, through the localization of the autophagy receptor p62 (also known as SQSTM1), with the high transduction efficiency and minimal toxicity of BacMam 2.0 technology. To perform an image-based analysis for autophagy, simply add the BacMam 2.0 RFP-p62 reagent to your mammalian cells, incubate overnight to ensure adequate protein expression, and then visualize using standard RFP (red fluorescent protein) settings. The RFP gene included in this chimera is mKate2. This monomeric fluorescent protein is very bright, has excellent pH resistance, and improved photostability.
Each Premo™ Autophagy Sensor RFP-p62 Kit includes BacMam 2.0 RFP-p62 reagent and chloroquine diphosphate, which is used to inhibit autophagy.
This RFP-p62 chimera is recommended for use with green-emitting fluorescent proteins or dyes.
• Highly efficient delivery system: >90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
• Fast and convenient: simply add the Premo™ Autophagy reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
• Robust: the Premo™ Autophagy reagents are non-replicating in mammalian cells, lack observable cytopathic effect, and are suitable for biosafety level (BSL) 1
• Multiplex-enabled: additional BacMam or fluorescent reagents can be used in conjugation with the Premo™ Autophagy products to detect and monitor additional targets of interest
The p62 protein, also known as sequestosome (SQSTM1), is an ubitiquitin-binding protein that functions as a receptor for cargos destined to be degraded by the cellular autophagic machinery. When autophagy is induced the p62 protein localizes to the autophagosomes and is subsequently degraded. Conversely, with the inhibition of autophagy, the p62 protein accumulates in the autophagosome. Thus, the subcellular localization a p62-fluorescent protein chimera serves as a useful marker for the induction and inhibition of autophagy.
To ensure reliable, high levels of expression in a wide variety of mammalian cells, the Premo™ Autophagy Sensors utilize BacMam 2.0 technology. BacMam reagents (insect Baculovirus with a Mammalian promoter) do not replicate in mammalian cells, are non-cytoxic (biosafety level 1), and are ready to use. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high co-transduction efficiency; therefore, multiple BacMam reagents can be used in the same cell.
Each Premo™ Autophagy Sensor RFP-p62 Kit includes a vial of chloroquine diphosphate. Chloroquine has been demonstrated to inhibit autophagy by elevating lysosomal pH and therefore inhibiting the fusion of autophagosomes with lysosomes and preventing the subsequent lysosomal protein degradation.