Thermo Scientific™

Sulfo-SBED Biotin Label Transfer Kit - Western Blot Application

Catalog number:  33073

Related applications:

Protein Biology

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Description

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.

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). 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.

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.

Related Products
Sulfo-SBED Biotin Label Transfer Reagent

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Product Line: Pierce™
Crosslinker Type: Heterotrifunctional
Labeling Method: Chemical Labeling, Label Transfer
Reactive Moiety: Sulfo-NHS Ester, Aryl Azide
Solubility: Water
Spacer: Cleavable, Long

Contents & storage

Sufficient For: 8 label transfer reactions for subsequent western blot analysis
• Sulfo-SBED, 8 x 1 mg
• PBS pack (makes 500 mL), 1 pack
• Label Transfer Buffer (20X), 200 mL
• Streptavidin-HRP Conjugate, 0.1 mg
• Dithiothreitol (DTT), 8 x 7.7 mg
• Slide-A-Lyzer MINI Dialysis Devices Plus Float, 10K MWCO, 10-device kit

Store at 4°C