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.

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.

优点：

数据：

P62 accumulates in LC3B positive vesicles upon inhibition of autophagy.

U2-OS cells were transduced with either Premo™ Autophagy Sensor GFP-p62 or Premo™ Autophagy Sensor RFP-p62 and cultured for 24hours. Cells were then treated with either vehicle (water) or 60uM Chloroquine for a further 16 hours. Cells were then processed for ICC and stained with anti-LC3B (detected using a goat anti-rabbit Alexa Fluor® 488 or 594 secondary antibody) Cells were counterstained with Hoechst 33342 and imaged using standard DAPI/FITC/Texas Red filter sets. The number of P62-positive spots were counted for each condition and each Premo™ Autophagy Sensor for 20 cells from two separate experiments.

Schematic depiction of the autophagy pathway including P62 and LC3B

The first step involves the formation and elongation of the isolation membranes or phagophore. The second step entails the expansion and sequestering of the cytoplasm and formation of the double-membrane autophagosome. During this step the LC3 protein is recruited to the auophagosome and P62 along with the attached ubiquitin cargo attach to the LC3B protein. The fusion of lysosome with autophagosome generate the autolysosome. At this step the LC3B disassociates and the P62 and the attached ubquitin cargo are degraded.

相关产品：

▪Premo™ Autophagy Sensor RFP-p62 Kit（货号#P36241）

▪Premo™ Autophagy Sensor GFP-p62 Kit（货号#P36240）

▪Premo™ Autophagy Sensor GFP-LC3B Kit（货号#P36235）

▪Premo™ Autophagy Sensor RFP-LC3B Kit（货号#P36236）

▪Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit（货号#P36239）