The Premo™ Autophagy Sensor GFP-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 GFP-p62 reagent to your mammalian cells, incubate overnight to ensure adequate protein expression, and then visualize using standard GFP (green fluorescent protein) settings. The GFP gene included in this chimera is TagGFP2, which has been demonstrated to mature 1.6-times faster than TagGFP and has increased pH stability when compared to Emerald GFP.

Each Premo™ Autophagy Sensor GFP-p62 Kit includes BacMam 2.0 GFP-p62 reagent and chloroquine diphosphate, which is used to inhibit autophagy.

This GFP-p62 chimera is recommended for use with red-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 GFP-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：

Imaging motility of P62-positive vesicles along microtubules.

A549 cells were transduced with Premo™ Autophagy Sensor GFP-p62 and CellLight® Tubulin-RFP and cultured for 24 hours. Chloroquine at a final concentration of 60uM was added and the cells were cultured for a further 16 hours. A549 cells were labeled with Hoechst 33342 and imaged using standard DAPI/FITC/TRITC filter sets.

P62 accumulates in LC3B positive, acidic vesicles upon inhibition of autophagy.

A549 cells were transduced with Premo™ Autophagy Sensor GFP-p62 and Premo™ Autophagy Sensor LC3B-RFP and cultured for 24hours. Chloroquine at a final concentration of 60uM was added and the cells were cultured for a further 16 hours. A549 cells were labeled with 50nM LysoTracker® Deep Red * Hoechst 33342 and imaged using standard DAPI/FITC/TRITC & Cy5 filter sets.

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.

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）