Fig. 2.

Representative interaction experiments using Sulfo-SBED cross-linker. Sulfo-SBED reacted purified His₆-T₇-DmsD was incubated with wild-type soluble prey protein fraction (~1.6 μg bait to prey extract protein ratio) and exposed to UV to permit cross-linking. Parallel experiments performed with non-reacted control. Cross-linked samples were cleaved with DTT to transfer the biotin label to the interacting partner and purified on an avidin column. After incubation the various soluble cell extract samples were run on a 12% SDS-PAGE gel and electroblotted. Following blocking in 10% milk/TBS/0.2% azide, the blot was initially probed with 100 ng/mL Streptavidin–HRP (Novagen) prior to development with Opti-4CN HRP developer reagent (Bio-Rad). Therefore bands in the figure contain biotin. Lane 1, Sulfo-SBED cross-linker reacted His₆-T₇-DmsD with soluble cell extract; lane 2, His₆-T₇-DmsD with no cross-linker exposed to cell extract; lane 3, no His₆-T₇-DmsD added to the cell extract. Molecular masses (in kDa) are indicated to the left. Non cross-linked biotin labeled DmsD bands are highlighted with an arrow indicating both monomer and dimer forms (confirmed by anti-DmsD antibody). Additional bands found in control lanes 2 and 3 are naturally occurring biotinylated or biotin-binding proteins.