Covalent protein modification by TCEP?

Dear All,

has anyone ever notice an impact of TCEP on total mass of the target protein? We have some strange effects, never observed before.

Best

Sabine

Posted on 12-Jun-2014 12:21 CEST

Hi Sabine,

no, we have never observed anything like this, despite exclusively using TCEP as reducing agent in our protein isolations. We do also regularly check Mw by LC-(ESI)-MS, in particular after protease digest of fusion proteins, but what you describe has never come to our attention.

I would however not generally rule it out, though one of the reasons for us to swap to TCEP (from ├čmEtOH and DTT) particularly was our concern about potential reactivity of other reducing agents. There are some reviews that describe reactions between active site cystines/methionines and reducing agents, which leads to the generation of H2O2 and thereby to e.g. generation of artifacts in enzyme assays, but this is quite differnt from covalent modification by a phosphine that you suppose.

Anyone else with more useful information?

 

Cheers,

Tim

 

Posted on 12-Jun-2014 13:40 CEST

Dear Tim,

thanks for the reply. We also prefer TCEP, as it is not forming any adducts. I guess the observed mass difference must have other reasons.

We never use bME, because it forms mixed disulfides with the proteins. This can also happen with DTT , but less frequently.

Best

Sabine

Posted on 12-Jun-2014 16:16 CEST
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