Someone else recently asked the same question. One answer was to see if the protein runs at different rates on SDS-PAGE when reduced or not reduced. Another answer was to selectively label disulfide bonds with a fluorescent dye. This is done as follows: First, block free thiols on the protein with N-ethylmaleimide and remove the excess reagent. Then reduce half the protein with TCEP, leaving the other half unreduced.  React both samples with fluorescein maleimide. Run the samples on SDS-PAGE and test for fluorescence. If the protein has a disulfide bond, the reduced sample will be fluorescent. (The unreduced sample is a negative control that should not be fluorescent).

Someone else recently asked the same question. One answer was to see if the protein runs at different rates on SDS-PAGE when reduced or not reduced. Another answer was to selectively label disulfide bonds with a fluorescent dye. This is done as follows: First, block free thiols on the protein with N-ethylmaleimide and remove the excess reagent. Then reduce half the protein with TCEP, leaving the other half unreduced.  React both samples with fluorescein maleimide. Run the samples on SDS-PAGE and test for fluorescence. If the protein has a disulfide bond, the reduced sample will be fluorescent. (The unreduced sample is a negative control that should not be fluorescent).

Hi,

running SDS-PAGE with and without reducing agent could show the difference. Note, that this is not sufficient to conclude that the protein had a disulfide bond in its native state. It could be formed upon denaturation/unfolding under oxidizing conditions in the sample.

If you know where the protein is localized in the cell it could help too. Secreted proteins are more likely to have disulfide bonds.

My favourite method would be mass spec of the intact (purified) protein to see if you have -2 Da for each potential disulfide bond. This usually is fast and accurate enough nowadays (if you have the Mass Spectrometer). If you get 2x the mass of the protein with -2 it could have an intermolecular disulfide bond.

The best would be crystallizing and looking at the bonds directly if possible, but that's obviously not the easiest way.

Thanks Adam. I can use your approach. Thanks Huseyin. I am doing this as a part of a small project. So, MS and crystallization will not suit me. But thanks as I can use your suggestion later in my research life.

Thanks all.