Dear Angelo

theoretically the best way to maintain the S-S reduced after DTT removal is perform a buffer exchange (eg with PD-10 desalting column) to remove the DTT inside glovebox chamber under nitrogen atmosphere using a buffer degassed with nitrogen flux. When i was phd i performed this protocol several time to produce reduced apo-human Sco1 and apo- Human cox17 able to bind copper (I) or to perform cisteine alkylation with AMS.

However to my knowledge, a more simple way to acheive the same result in your case is replace the DTT with the TCEP, with is compatible with cysteine alkylation with malelmmide, and therefore you dont 'have the issue to remove it before alkylation but you can do it, at the air by desalting or dialysis after alkylation.

Article Characterization of Reactions between Water-Soluble Trialkyl...

good luck

Manuele

We use TCEP for the reduction and add the maleimide moiety, no need to remove the TCEP (or you can use immobilized TCEP resin)

Dear Angelo Monteiro thank you for your interesting technical question. This has been the subject of an earlier discussion on RG. Please have a look at the answers given to the following closely related question:

Protocol for tcep and maleimide reaction?

https://www.researchgate.net/post/Protocol-for-tcep-and-maleimide-reaction

(10 answers)

Also please see the following relevant article:

A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry

https://proteinark.com/Literature/TCEP%20vs%20DTT%20publication.pdf

This article can be freely downloaded as pdf file.

Unfortunately, I can't use any phosphines as it will most probably reduce other moieties (such as azide), which I want to keep.