The ProteoTuner Shield System C 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 pPTunerC 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.
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 showed a better destabilization profile when the DD tag was fused to the N-terminus of the protein of interest (Systems N). Specific DD tag mutants for C-terminal tagging are available as well (System C); however they have a slightly reduced destabilization activity in the absence of the Shield1 ligand.
Applications
- Protein function in pathways
- Functional analysis of subunits of a protein complex
- Functional analysis of essential proteins
