| Products/Services Used | Details | Operation |
|---|---|---|
| Bacterial Expression System | All cytosolic sensors were constructed with a yeast codon-optimized roGFP2 moiety (Genscript) as described previously21 . For the roGFP2-Prx2 construct the human Prx2 sequence was synthesized with codons optimized for yeast expression (Genscript)...HyPer was synthesized with codons optimized for yeast expression (Genscript) and subcloned in the p415-TEF vector...RoGFPTsa2ΔCR was synthesized with codons optimized for E. coli expression (Genscript) and subcloned in the pDEST17 vector with an N-terminal His6 tag. | Get A Quote |
Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are ∼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expres... More
