Dear Deepak Deepak ,

The precise concentrations and circumstances will of course vary upon the different proteins studied, but perhaps you find an answer in the following paper:

Article Oxetane Grafts Installed Site-Selectively on Native Disulfid...

Here TCEP is used and you find (page 37 and further) in the enclosed supplement more details about the procedure.

Hope this helps you a bit further.

Best regards.

Dear Deepak

Concentration of reducing agent, it depends also from the concentration of your protein sample.

Generally you need to add a a large excess of the reducing agent and incubate a little to let time to work. DTT and TCEP are both quite strong and similar. I think that incubation with 10mM of both will be able to fully reduce your protein.

i did something similar many times in the past to prepare Cu(I) metallated protein.

The best approach is:

1) reduce the sample

2) buffer exchange it readily by desalting to remove the reducing agent

3) add the cysteine chelating agent.

theoretically if you would like to be sure that 100% of cysteines are still reduced when you add the chelating agent, you need to

- perform all the step (dissolution of reducing agents,, addiction of it to the sample, buffer exchange and addition of cysteine chelating agent) inside an glove box chamber with a nitrogen atmosfere (no oxigen)

- degas all the buffers with nitrogen flux.

In case you do not have it, you have to:

- degas all the buffers with nitrogen flux

- minimize the timeline of the desalting and addition of chelating agent steps tand work with diluted protein samples (10-20uM maximun) to reduce the probability and kinetics of intermolecular S-S reformation.

In this way is possible that you will not obtain 100% of the desidered product, but in case. once the protein is stable after cisteine chelation, you can remove the dimeric unwanted fraction with a SEC coloumn.

good luck

Manuele

If you use TCEP as the reducing agent, you can perform a tethering reaction with maleimide chemistry without first removing the TCEP, so that the disulfide bond will stay reduced.

If you use DTT, you will have to remove it first. The disulfide bond will start to reform as soon as the DTT is removed (as long as oxygen is present). It will be difficult to remove the DTT quickly and completely from such a small protein.

Deepak - please also see this potentially useful article entitled "Reagents for rapid reduction of disulfide bonds in proteins":

Article Reagents for rapid reduction of disulfide bonds in proteins

According to tjhis article "Dithiothreitol (DTT) is the most popular disulfide-reducing reagent. A comparison of reactivities of bis(2-mercaptoethyl)sulfone (BMS), [N,N’-dimethyl-N,N’-bis(mercaptoacetyl)hydrazine (DMH), and DTT toward disulfide groups in several proteins under non-denaturing conditions at pH 7 is made. Both BMS and DMH reduce disulfide bonds in proteins at pH 7 faster than DTT does by a factor of ∼5-7 in non-denaturing conditions. BMS is, therefore, more reducing than DMH and slightly less reducing than DTT. All these dithiols (BMS, DMH, and DTT) have significantly high reduction potentials and reduce noncyclic disulfides completely."

There is another interesting article entitled "A pratical protocol for the reduction of disulfide bonds in proteins prior to analysis by mass spectrometry". In this study the authors also use DTT as reducing agent. This paper is available as public full text on RG:

Article A Practical Protocol for the Reduction of Disulfide Bonds in...

Also please not that there is a closely related thread on RG which could give you some additional answers:

https://www.researchgate.net/post/Does-anyone-know-how-long-a-disulfide-bond-in-a-protein-remains-reduced

Hi,

Thanks to all for the answers, I reduced the protein successfully and stays reduced for long enough.

Which column do you recommend to remove DTT from reduced protein?

PD10 or NAP 5 columns or are there other better options?