I am working with a system in which my protein needs to have a disulfide reducer like tris(2-carboxyethyl)phosphine (TCEP) present, but the binding targets I'm studying have a nitroxide free-radical spin label on them (PROXYL; similar to MTSL). Will TCEP reduce the nitroxide free radical? (I know sodium ascorbate does).
It is very likely that it will be reduced.
The real question is how fast. Some nitroxides are more resistant to reduction than other. Also you can try to lower the concentration of the reducing agent to buy time.
Philippe Mellet Thank you for the response, Philippe. I am using 1 mM TCEP at present. I am unsure how fast it is being reduced, although some of the paramagnetic species appears to be reduced to the diamagnetic species within an hour or two based on NMR spectra. That being said, I do not know if it is a TCEP-related issue or something else. I suppose I'll have to do time-dependent NMR studies. It's important that all of the species be in the paramagnetic form, though, because I'm doing paramagnetic relaxation enhancement studies.