How to Determine Disulfide Bonds

Biochemistry, Molecular Biology, and Cell Biology Protocols >> How to determine disulfide bonds

1. Trypsinize the protein in its native structure, and determine the masses of the peptide fragments; sequence each peptide. Reduce the nonreduced peptides, and they should produce the masses of each individual peptide. Can also try to use other enzymes besides trypsin.
2. Another way is to solve the 3-dimensional structure, such as by X-ray crystallography.
3. Another way is by site-directed mutagenesis. By mutating the two cysteines that you think may be forming a disulfide bond, and observing its migration on nonreducing SDS-PAGE before and after reduction, the potential pair of disulfide bonds can be determined. If there is a change in migration pattern, there may be a disulfide bond present.
4. Another way is via molecular modelling. If a structure of a related protein has been solved, a molecular model of a related protein can be generated. If the cysteines are sufficiently close to each other, a disulfide bond may be possible.