A powerful tool for tagging and discovering protein interaction partners.
The Thermo Scientific Pierce Sulfo-SBED Biotin Label Transfer Reagent is a multifunctional reagent for labeling a purified protein and then covalently transferring the attached biotin tag onto specific interactors of that protein.
Label Transfer is a powerful in vitro method for protein interaction discovery. A growing number of publications feature the use of Sulfo-SBED Biotin Label Transfer Reagent to identify previously unknown protein interaction binding partners and to more fully characterize the specific protein binding domains of other protein interactions (see cited references below).
What is Sulfo-SBED?
Sulfo-SBED is the abbreviation for Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3'-dithioproprionate. It is a heterobifunctional chemical crosslinker capable of covalently attaching to primary amines at one end and to nearly any protein functional group at the other end. Unlike typical crosslinkers, Sulfo-SBED also includes a biotin group and a cleavable disulfide spacer arm. Together these features allow one to sequentially crosslink interacting proteins and transfer the biotin affinity tag from one protein (i.e., a purified "bait" protein) to another (possibly unknown "prey" protein).
Structural and functional properties of Sulfo-SBED for biotin label transfer:
Amine-reactive NHS-ester group – for labeling a purified "bait" protein at the N-terminus and side chain of lysine residues
UV light-activatable aryl azide group – for crosslinking nonspecifically to the protein side chains and backbone of the interacting protein after allowing protein binding to occur
Cleavable disulfide bond (S-S) – can be reduced to release the crosslinker from the original "bait" protein
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.
Typical protocol for performing a label transfer experiment with Sulfo-SBED:
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 inteactors ("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 (Figure 1).
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.
Figure 1. Experimental strategy for Sulfo-SBED biotin label transfer and analysis by Western blotting.
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Sulfo-SBED Biotin Label Transfer Kit - Western Blot Application
Formulation: Multi-component kit with Sulfo-SBED reagent Sufficient For: 8 label transfer reactions for subsequent Western blot analysis
Kit Contents: Sulfo-SBED, 8 x 1mg PBS pack (makes 500mL), 1 pack Label Transfer Buffer (20X), 200mL Streptavidin-HRP Conjugate, 0.1mg Dithiothreitol (DTT), 8 x 7.7mg Slide-A-Lyzer MINI Dialysis Devices Plus Float, 10K MWCO, 10-device kit
Sulfo SBED Biotin Label Transfer Reagent
Formulation: Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3'-dithioproprionate; powder solid Sufficient For: 23mL of typical 1X (0.5mM) reaction solution, or approx. 5 uses at 2mg reagent per use
Sulfo-SBED Biotin Label Transfer Reagent, No-Weigh Format
Formulation: Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3'-dithioproprionate; powder solid Sufficient For: 18mL of typical 1X (0.5mM) reaction solution (total), or 8 separate uses