Here's one method:

First, treat the unreduced protein with N-ethylmaleimide (NEM) to block any cysteine that is not in a disulfide bond. Remove excess NEM (you can use a gel filtration spin column). Add TCEP reducing agent to the protein to reduce the disulfide bond if there is one. React the protein with fluorescein maleimide. Run the protein on SDS-PAGE. If there was a disulfide bond, the protein will be fluorescent. As a negative control, omit the TCEP reduction.

@Adam B Shapiro

Thanks for your answer. I just have 3 questions :

1- the fluorescent protein will be detected visually without using any other machine ?

2- will just running the  protein in reducing and  non reducing conditions in SDS-PAGE tell me if it has a disulphide bond or not?

3- what gel filtration spin column do you recommend to use?

Thanks again for your answer 

Hi there,

Running SDS PAGE with reduced and non reduced samples in sometimes successfull : it depends on the impact of the presence of  the disulphide bridges on the overall architecture of the denatured protein... If there is an impact, the reduced form of the protein will run faster than the non reduced one.

You can visualize the fluorescence using a UV transilluminator.

I often use pre-packed Bio-Rad micro bio-spin columns.

Adam outlined a very nice method for visualizing the presence of disulfides, although I would add that if your disulfides are intramolecular, TCEP might not be able to reduce these due to its bulky nature, ergo, it would be more reasonable to carry out the reduction using mercaptans (like DTT).

In our lab, we routinely use mass spectrometry to check for disulfides. The principle is very simple: One sample is reduced with DTT and another one remains unreduced. Then we run these through a C4 column in acidic conditions using an UPLC system and measure the masses (we use ESI-TOF MS for this). From the chromatogram you will already be able to see if something happens in terms of equilibrium or sample homogeneity, although I don't trust this quantitatively like I trust gel filtration. From the masses you get, you will easily see if your protein is reduced from a dimer to a monomer, for instance, or if a monomer with an intramolecular disulfide is reduced (mass increases by 2 Da, due to the two extra hydrogens when -S-S- becomes 2 x -SH). Mass spec is great because you need very little sample and when using an LC step the sample doesn't need to be exactly pure. One should also remember that especially intermolecular disulfides are rather common artifacts arising in recombinant protein preparations without reducing agents.

Hope this helps,

Arne

Thank you all for your kind answers . I really appreciate it

DTNB assay is the best. Please Google it and you will find it. If there is a free thiol, it will detect it. 

DTNB is not a very sensitive method for detection of thiols, requiring several micromolar., which might be a limitation for a protein. Thio-Glo is a fluorogenic thiol detection reagent that is more sensitive.