Retro-X ProteoTuner Shield System N
Retro-X ProteoTuner Shield System N
Brand: Takara Bio.
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Retro-X ProteoTuner Shield System N
Direct manipulation of the presence and absence of a specific protein of interest is a very powerful tool for analyzing protein function. The Retro-X ProteoTuner Shield System N uses a unique method to regulate the amount of protein of interest present in a cell, quickly and directly. It utilizes a ligand-dependent destabilization domain (DD) and its membrane permeable stabilizing ligand, Shield1. The DD is based on a 12 kDa mutant of the FKBP protein, which is expressed as a tag onto the N-terminus of your protein of interest cloned into the pRetroX-PTuner Vector. In the presence of Shield1, the DD-tagged protein is stabilized and will accumulate 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. However, in the absence of the protective ligand Shield1, the DD-tagged protein of interest is rapidly degraded. Removing the Shield1 (by splitting the cells into media without Shield1) allows for protein destabilization, causing a fast degradation of the protein of interest. The extent of stabilization via Shield1 directly correlates with the amount of the ligand in the medium, so it is possible to tune the amount of protein of interest present in the cell, by controlling the amount of the Shield1 ligand.
Overview
- Rapid kinetics: protein level changes in minutes allows accurate functional analysis.
- Precise tuning: precise control of protein level by controlling the dose of Shield1.
- Reversible control: "protein on" to "protein off" for convincing gene-function studies.
- What you get: each kit is supplied with a plasmid vector and an aliquot of Shield1.
- NOTE: Most of the proteins that we tested show a better destabilization profile when the DD tag is fused to the N-terminus of the protein of interest (N Systems). Specific DD tag mutants for C-terminal tagging are available as well (C System); however, they have a slightly reduced destabilization activity in the absence of the Shield1 ligand.
- For retrovirus production, use in combination with our Retro-X Universal Packaging System.
- For lentivirus production, use in combination with our Lenti-X Packaging Single Shots.
Applications
- Protein function in pathways
- Functional analysis of subunits of a protein complex
- Functional analysis of essential proteins
Dose-dependent stabilization of DD-tagged AcGFP1 fluorescent protein
Dose-dependent stabilization of DD-tagged AcGFP1 fluorescent protein. Cells were transfected with pDD-AcGFP1-PL, treated with the indicated amounts of Shield1, and the amount of stabilized DD-AcGFP1-PL was quantified by Western blot using the Living Colors A.v. Monoclonal Antibody(JL-8) to detect AcGFP1.
Ligand-dependent, targeted and reversible protein stabilization
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.
Protein stabilty controlled using the ProteoTuner shield system
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 5–8: 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.
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Dose-dependent stabilization of DD-tagged AcGFP1 fluorescent protein
Dose-dependent stabilization of DD-tagged AcGFP1 fluorescent protein. Cells were transfected with pDD-AcGFP1-PL, treated with the indicated amounts of Shield1, and the amount of stabilized DD-AcGFP1-PL was quantified by Western blot using the Living Colors A.v. Monoclonal Antibody(JL-8) to detect AcGFP1.
Ligand-dependent, targeted and reversible protein stabilization
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.
Protein stabilty controlled using the ProteoTuner shield system
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 5–8: 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.
