Hi! I study the effect of formulation excipients on protein degradation pathways and was looking disulfide-mediated aggregation by comparing reduced/non-reduced SDS page samples. I am using TCEP as my reducing agent. I noticed that in every reduced sample (lane closest to ladder (left), lane furthest from ladder (right) is exactly the same but WITHOUT reducing agent) I detect a new band between 20-25kDa. My protein of interest is between 45-50 kDa. I also see the supposed dimer around 90kDa disappear in the TCEP reduced samples which is what I would expect. I initially suspected that the emergent band could be the result of TCEP hydrolysing the peptide bond of my protein somehow but don't see this being reported in the literature. I analysed the reduced and non-reduced samples via LCMS and oddly don't see much difference between them. Any input would be greatly appreciated!!
This is likely a question of protein conformation. -S-S bridges hold the protein in a more compact conformation than the fully reduced protein will assume because of Coulomb repulsion between bound SDS molecules. That is why proteins are usually fully reduced before electrophoresis by heating them with ßME or (better) DTT.
A new band between 20-25kDa suggests that your protein in TCEP (reduced) degraded compared to non-reduced environment where it was compact/well folded (presence of band around 90 kDa in non-redued sample). The effect may be due to absence of disulfide bridge, making protein susceptible to degradation.
I agree with Englebert's explanation, disulfide bonds often make proteins more compact and mobile in SDS-PAGE. I would caution that reducing agents do not "stay put" in PAGE gels; it can be quite confusing as they can reduce adjacent bands to various extent as they travel (as a function of conc. and some other variables). For that reason I always include a few intervening wells between +/- reducing agents. Light scatter or sedimentation might give you more hints as to conformational changes associated with S-S bonds and degradation mechanisms, good luck!
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