Typical protocol for label transfer experiment:
• Add a few microliters of dissolved Sulfo-SBED Reagent to 0.5-1 mL of purified bait protein in PBS.
• Incubate mixture for 30-120 minutes on ice or at room temperature in the dark.
• Desalt or dialyze (in subdued light) to remove excess non-reacted Sulfo-SBED from the labeled bait protein.
• Add labeled bait protein to cell lysate or other solution containing putative target protein interactors ('prey').
• When interaction complexes have formed, expose the solution to ultraviolet light (365 nm) for several minutes.
• Analyze products by one of several methods:
• Western Blotting: Cleave crosslinks in DTT, separate proteins by SDS-PAGE, and detect biotinylated bands by Western blotting with streptavidin-HRP.
• Purification and Mass Spec or Sequencing: Affinity-purify biotinylated proteins or peptide fragments following trypsin digestion and perform MS or sequencing to characterize the proteins involved.
Sulfo-SBED Biotin Label Transfer Kit - Western Blot Application