Crosslinkers sulfo-SBED

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

MTS-ATF-biotin and MTS-ATF-LC-biotin are trifunctional crosslinkers similar in design to sulfo-SBED discussed previously, but in addition to the biotin handle, they contain a... 

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but instead of transferring a radioactive label, it contains a fluorescent portion that is transferred to a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein interaction. Cleavage of a disulfide bridge after capture of interacting proteins results in transfer of a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modification then can be used to detect or isolate the unknown interactor for subsequent identification. 

Bait proteins modified with Sulfo-SBED may precipitate if the level of modification is too high, primarily due to the hydrophobic nature of the crosslinker and the biotin handle. To prevent precipitation or at least minimize it, adjust the molar excess of Sulfo-SBED over the bait protein to a level where the protein remains in solution. Some precipitation may be removed by centrifugation or filtration prior to use. 

Figure 28.12 Sulfo-SBED first is used to label a bait protein through reaction of the sulfo-NHS ester with available amine groups on the protein, yielding an amide bond linkage. This labeled bait protein then is added to a sample containing proteins that potentially could interact with the bait. After an incubation period, the sample is exposed to UV light to photoactivate the phenyl azide group. This reaction causes any interacting prey proteins to be crosslinked with the bait protein, forming a complex containing a biotin affinity tag.

Add a quantity of the Sulfo-SBED solution to the bait protein solution so that a 1- to 5-fold molar excess of crosslinker over the bait protein results in the reaction mixture. Mix well. Using greater quantities of Sulfo-SBED to the bait protein may result in precipitation due to the hydrophobic nature of crosslinker. In addition, over modification of the bait protein with the crosslinker may block sites of protein interaction, thus preventing complex formation. As a practical example, Horney et al. (2001), used a 1 1 molar ratio of Sulfo-SBED to the bait protein IGF-1 with success. 

---