Lenti-XTM ProteoTunerTM Shield System C

Description

The Lenti-X ProteoTuner Shield System C provides a unique method for quickly and directly regulating the amount of your protein of interest present in a cell, and combines it with versatile lentiviral delivery. The system utilizes a ligand-dependent destabilization domain DD-C and a membrane-permeant stabilizing ligand, Shield1, to quickly and directly regulate the amount of your protein of interest present in a cell or organism. First, your gene of interest is cloned into the pLVX-PTunerC Vector, and expressed as a fusion with the destabilization domain DD-C . Then the Shield1 ligand is either added or withheld, depending on your experiment. In the presence of Shield1, the DD-C-tagged protein is stabilized and accumulates inside the cell. This ligand-dependent stabilization occurs very quickly, and has been observed as soon as 15-30 minutes after the addition of Shield1. In the absence of Shield1, the DD-C-tagged protein of interest is unstable. Consequently, removal of Shield1 allows for active and controllable degradation of the protein of interest. The extent of stabilization via Shield1 can be adjusted, and directly correlates with the amount of the ligand in the medium. Therefore, you can tune the level of your protein of interest present in the cell, by controlling the amount of the Shield1 ligand. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back Required Products The Shield1 compound stabilizes a protein of interest tagged with the destabilization domain DD , which is based on a mutated version of the FKBP protein. It is used to protect the DD fusion protein of interest from proteasomal degradation. The amount of Shield1 can be varied to tune the amount of stabilized protein of interest within the cell. Back Ligand-dependent, targeted and reversible protein stabilization.A small destabilization domain DD; blue is fused to a target protein of interest. The small membrane-permeable ligand Shield1 red binds to the DD and protects it from proteasomal degradation. Removal of Shield1 however, causes rapid degradation of the entire fusion protein. The default pathway for the ProteoTuner systems is degradation of the fusion protein unless Shield1 is present. Back Protein stabilty controlled using the ProteoTuner shield system.DsRed-Express was cloned into pRetroX-PTuner IRES and used to infect HeLa cells. The amount of DD-tagged DsRed-Express stabilized by different concentrations of Shield1 was detected via Western blot using the Living Colors DsRed Polyclonal Antibody. Lane 1: molecular weight marker. Lane 2: 1X loading buffer. Lane 3: untreated HeLa cells no virus, no Shield1 . Lane 4: HeLa cells infected with the DD-DsRed Express construct; no Shield1. Lanes 58: HeLa cells infected with the DD-DsRed-Express construct and treated with 50, 250, 500, and 1,000 nM Shield1 respectively. Lane 9: 1X loading buffer. Lane 10: HEK 293 DsRed-Express stable cell line. Back Control of actin stability with the ProteoTuner system.In the absence of Shield1, no DD-AcGFP1 is observed Panel B despite the presence of a normal actin filament network Panel A . In the presence of Shield1, DD-AcGFP1 is stable and present in the actin network Panel D . Back DD fusions exhibit predictable, dose-dependent protein stabilization by Shield1; using the DD optimal for your protein is crucial in getting the best performance.While DD-N solid blue line is suitable for N-terminal fusions and even C-terminal fusions for many proteins, DD-C dashed blue line is optimal for C-terminal fusions and for those proteins that do not tolerate N-terminal fusions. HEK 293 cells expressing the DD fusions noted in the figure legend were treated with varying concentrations of Shield1. 12 hr later, the amount of AcGPF1 stabilized by different concentrations of Shield1 was detected by fluorescence intensity using flow cytometry. DD-C at the C-terminus of AcGFP showed a lower basal level of protein in the absence of Shield1 purple line; see inset figure . MFI = mean fluorescence intensity. Back Back