I have a few proteins that I produce recombinantly in E. coli cells. All of them are disordered proteins and are stored in the presence of 4mM DTT at -20°C before use. One of them also contains 1.5M urea in the storage buffer. I have found that over time, the concentration of the proteins, measured at 280nm, increases significantly. Is this due to the presence of DTT? I know that oxidised DTT absorbs strongly around 280nm, but my blank also contains DTT. How can I resolve this issue?
You should be able to distinguish between an increase in the protein concentration due to sublimation of the liquid versus accumulation of oxidized DTT by performing a different type of protein assay. The Bradford assay may be suitable for your samples, since it is not affected by DTT.
If you have access to a -80 freezer, I recommend that in preference to -20 for long-term protein storage. The lower temperature prevents sublimation.
I agree with Prof. Adam. Sublimation can cause increase in protein concentration.
I also think it might be due to the precipitation/aggregation. Since they are disordered proteins, freezing them at -20C slowly may encourage nucleation and precipitation/aggregation. This is one of the reason, proteins are flash frozen, to prevent precipitation/aggregation.
Another reason could be the presence of urea or DTT. Urea and DTT are chaotropic agents (cause protein to unfold) and usually causes decrease in fluorescence emission intensity in relation to increasing concentrations of the agents. However, there are few proteins that behave contradictorily where fluorescence intensity increases with increasing concentrations. The reason for the increase in intensity is ambiguous. That said, the absorption/emission correlation can be used to understand the change in concentration.
To resolve this, I would suggest checking for change in fluorescence emission with increase in urea/dtt concentration. If you see an increase (and not a decrease like usual), then that should answer the question. You can also try adding glycerol or PEG to see if protein precipitation/aggregation was the reason.
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