Sulfo-SBED Biotin Label Transfer Reagent, 10 mg - FAQs

What makes Sulfo-SBED Biotin Label Transfer Reagent useful for investigation of protein interactions?

Sulfo-SBED allows one to transfer a biotin tag from an amine-containing protein to a second protein (reacted with the phenyl azide). The amine-containing protein can be removed by reduction of the disulfide in the Sulfo-SBED spacer arm. This leaves just the second biotinylated protein for detection.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Should the Sulfo-SBED coupling reaction be protected from light?

During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide functional group.

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Which reaction should take place first during Sulfo-SBED crosslinking?

The NHS-ester end of the crosslinker is subject to hydrolysis, therefore the amine-containing protein should be conjugated prior to the photoactivatable (aryl azide) reaction.

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What is the appropriate wavelength for the conjugation of the photoactivatable group in Sulfo-SBED Biotin Label Transfer Reagent ?

UV light at wavelengths greater than 300 nm are appropriate for conjugation.

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Is the Sulfo-SBED crosslinker soluble in organic solutions as well as aqueous solutions?

Yes, Sulfo-SBED is soluble in aqueous as well as organic solutions. It is most soluble in organics such as DMF and DMSO.

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Can Sulfo-SBED Biotin Label Transfer Reagent be stored in a stock solution?

No, the crosslinker is moisture sensitive. The sulfo-NHS ester is easily hydrolyzed.

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Can you provide the shelf-life for Sulfo-SBED Biotin Label Transfer Reagent?

Sulfo-SBED Biotin Label Transfer Reagent is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Can you provide the shelf-life for the Sulfo-SBED Label Transfer Reagent?

The Sulfo-SBED Biotin Label Transfer Reagent is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Terms-and-Conditions-of-Sale.pdf) for more details.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

What crosslinkers do you recommend to study protein-protein interactions that are mass spectrometry - compatible?

We offer Sufo-SBED (Cat. Nos. 33033 and A39260).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

What can I do if after label transfer of my sample with sulfo-SBED, I do not detect any signal in western blot?

1. Determine the label incorporation of the bait protein before photoactivation with the prey protein, by western blot analysis.
2. Check the UV light source to ensure good photoactivation (see Photolysis Information section of the Sulfo-SBED manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0011509_SulfoSBED_BiotinLabel_Xsfer_WestBlotApp_UG.pdf) for guidelines).
3. If the target (prey) protein concentration is too low, enrich the protein system if possible.
4. Optimize the antibody concentrations used in western blot.
5. It is possible that the protein interaction is blocking the epitope, which may occur with monoclonal antibodies and peptide-directed antibodies; in this case, use an antibody from a different source or probe with Streptavidin-HRP.
6. Milk contains endogenous biotin and its use as a blocking buffer during the detection assay could mask the bands of the biotin-labeled interaction partner.

During label transfer using the sulfo-SBED Biotin Label Transfer Reagent, how is the spacer arm cleaved to separate the bait protein from the label?

In the case of sulfo-SBED biotin, there is a disulfide in the spacer arm of this tri-functional crosslinker that is cleaved with reducing agents such as DTT, beta-ME or TCEP. As the disulfide lies between the contact point on the bait protein and the complex of the photoreactive group and biotin, reduction separates the bait protein from the prey protein; which is now biotin labeled.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

During label transfer using the sulfo-SBED Biotin Label Transfer Reagent, what groups are targeted on the bait and prey protein?

The NHS-ester of sulfo-SBED biotin will react with primary amines on the bait protein. The photoreactive aryl azide primarily targets nucleophiles, such as -NH2, -SH and -OH, but in their absence, it will react with any near neighbor molecule.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What are the label transfer reagents you offer?

Currently, we offer sulfo-SBED Biotin Label Transfer Reagent (Cat. Nos. 33033 and 33034) as well as the Sulfo-SBED Biotin Label Transfer Kit for Western Blot Application (Cat. No. 33073).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.