Proteasome Sensor Vector
Proteasome Sensor Vector
Brand: Takara Bio.
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Proteasome Sensor Vector
The Proteasome Sensor Vector (pZsProSensor-1) encodes the gene for Zoanthus sp. Green Fluorescent Protein (ZsGreen) fused to the mouse ornithine decarboxylase (MODC) degradation domain (amino acids 410–461). This vector is designed for studies of proteasome function in mammalian cells: Since the MODC degradation domain targets the constitutively-expressed protein for rapid degradation, the protein does not accumulate in cells until the proteasome is inhibited, which is indicated by an increase in green fluorescence. This vector also contains a kanamycin/neomycin selectable marker, as well as SV40 and pUC origins of replication.
Overview
- Easy fluorescent monitor of proteasome activity
- Compatible with individual cells or whole populations in real time
- Easy, non-invasive detection
- Simple and cost effective assay
- High-throughput compatible
Applications
- Drug screens
- Cancer studies
- Image-based HTS/HCS
- Real-time proteasome studies
Measuring proteasome activity in living cells
Measuring proteasome activity in living cells. HEK 293 cells stably transfected with the Proteasome Sensor Vector (Panel A) were selected in G418 and sorted by flow cytometry, and then treated with 10 μM ALLN for the indicated times. ALLN is a peptide aldehyde (Ac-Leu-Leu-Nle-al) that reversibly inhibits the proteasome’s chymotrypsin activity, preventing it from attacking the ZsProSensor-1 protein. As a result, ZsProSensor-1 quickly accumulates, resulting in a strong green emission signal that can be measured by fluorescence microscopy (Panel B), flow cytometry (Panel C), or fluorometry with a 96-well plate reader (Panel D).
High throughput screening (HTS) assay to identify proteasome inhibitors
High throughput screening (HTS) assay to identify proteasome inhibitors. Composite images of the HEK 293 ZsGreen Proteasome Sensor Cell Line were acquired using the ArrayScan HCS reader. The cells were stained with 10 μM Hoechst 33342 (blue). Panel A. The positive control bortezomib. Panel B. Disulfiram. Panel C. PDTC. Panel D. Negative control (PBS).
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Measuring proteasome activity in living cells
Measuring proteasome activity in living cells. HEK 293 cells stably transfected with the Proteasome Sensor Vector (Panel A) were selected in G418 and sorted by flow cytometry, and then treated with 10 μM ALLN for the indicated times. ALLN is a peptide aldehyde (Ac-Leu-Leu-Nle-al) that reversibly inhibits the proteasome’s chymotrypsin activity, preventing it from attacking the ZsProSensor-1 protein. As a result, ZsProSensor-1 quickly accumulates, resulting in a strong green emission signal that can be measured by fluorescence microscopy (Panel B), flow cytometry (Panel C), or fluorometry with a 96-well plate reader (Panel D).
High throughput screening (HTS) assay to identify proteasome inhibitors
High throughput screening (HTS) assay to identify proteasome inhibitors. Composite images of the HEK 293 ZsGreen Proteasome Sensor Cell Line were acquired using the ArrayScan HCS reader. The cells were stained with 10 μM Hoechst 33342 (blue). Panel A. The positive control bortezomib. Panel B. Disulfiram. Panel C. PDTC. Panel D. Negative control (PBS).
