Journal: Science Advances
Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems
Figure Lengend Snippet: Protein Persulfide Detection Protocol. Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.
Article Snippet: Biotinylated proteins were pulled down by streptavidin-coated magnetic beads (Sigma), and from this step, the same protocol was applied as for the purified protein samples, using 25 mM DTT instead of 5 mM TCEP to cleave the persulfidated proteins off the beads.
Techniques: Functional Assay, Affinity Purification, Magnetic Beads, Purification, Binding Assay