Reagents for biotin label transfer from bait protein -SH groups.

Biotin label transfer has emerged as a powerful new tool for protein interaction discovery and confirmation. Historically, the only reagents available were for transfer from primary amines (e.g., lysine side chains) on the bait protein to miscellaneous amino acid functional groups on the target prey protein. Sulfhydryl-reactive Mts-Atf-Biotin and its long chain analog, Mts-Atf-LC-Biotin, are exciting additions to the protein interaction tool chest by enabling biotin label transfer from sulfhydryl groups (cysteine side chains) on the bait protein.

Key structural and functional properties of Mts-Atf-Biotin Reagents for label transfer:

• Sulfhydryl-reactive methanethiosulfonate (Mts) group – for labeling a purified "bait" protein at the side chain of reduced cysteine residues, thereby temporarily attaching the biotin tag to the bait protein via a cleavable disulfide bond
• UV light-activatable perfluorophenylazide (Atf) group – for crosslinking nonspecifically to the protein side chains and backbone of the interacting protein after allowing protein binding to occur
• Biotin group – remains attached to target interacting protein after cleaving the disulfide bond, thereby tagging the previously unknown interacting protein(s) for affinity purification and detection.

How does biotin label transfer work?
The label-transfer method involves transfer of a biotin molecule from a purified bait protein to a binding partner designated as the prey protein. Using these reagents, a -SH-containing bait protein is first biotinylated via the Mts moiety. After removal of excess reagent and incubation in the dark with a putative prey protein or prey protein-containing lysate, the Atf moiety is activated with UV light capturing the interacting complex. Reduction of the disulfide bond formed by reaction of -SH with the Mts group of the label-transfer reagent results in the transfer of the biotin label from the bait protein to the prey protein. The biotin can be used to both enrich for and detect the interacting complex or prey protein using immobilized Monomeric Avidin or Streptavidin-HRP-based detection, respectively (Figure 1).



Figure 1. Label-transfer scheme. The bait protein is first biotinylated through the Mts moiety. An interacting prey protein is then captured by photoactivation of the Atf moiety. The interacting complex is isolated and the disulfide bond reduced, resulting in the biotin label being transferred to the interacting protein.

Related Products:

UV Lamp for Crosslinker Photoactivation
Monomeric Avidin Agarose Resin
Streptavidin-HRP
SuperSignal Chemiluminescent Substrates
DTT (Dithiothreitol), No-Weigh Format
Sulfo-SBED Biotin Label Transfer Reagent