Sulfo-SBED

Sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]-ethyl-1,3 -dithiopropionate MW 879.97 

The sulfo-NHS ester of sulfo-SBED is negatively charged and provides a degree of water solubility (about 5 mM maximum concentration) for the entire molecule. Limited water solubility is all that can be expected due to the large size of the trifunctional, most of it consisting of relatively hydrophobic structures. However, the reagent is much more soluble in organic solvents such asDMF (170 mM) andDMSO (125 mM). Concentrated stock solutions may be prepared in these solvents prior to addition of a small aliquot to an aqueous reaction mixture. 

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 min in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide functional group. The degree of derivatiza-tion should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

The following suggested protocol was developed by Barb Olson at Pierce Chemical for the labeling of soybean trypsin inhibitor with its subsequent complexation with trypsin. Modifications to this procedure may have to be done for other proteins. 

Dissolve 1.12 mg of sulfo-SBED in 25 xl of DMSO. Prepare fresh. 

ABNP is soluble in dimethylformamide (DMF) but insoluble directly in aqueous solution. Insulin labeling was done in DMF water at a ratio of 9 1. For molecules not soluble in organic solvent, such as proteins, the trifunctional first may be dissolved in DMF and a small aliquot added to an aqueous reaction medium. The nitrophenyl ester reactive group can be coupled to amine groups at alkaline pFI (7-9) and in buffers containing no extraneous amines (avoid Tris). Unfortunately, ABNP is not commercially available at the time of this writing. 

Additional information on the use of sulfo-SBED for the study of protein interactions can be found in Chapter 28, Section 3.1. 

Sample containing prey protein inleraclion partner. 

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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.

Mix the purified sulfo-SBED-modified STI with 5 mg of trypsin dissolved in 0.1M sodium phosphate, 0.15 M NaCl, pH 7.2. 

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... 

Although MTS-ATF-biotin and MTS-ATF-LC-biotin are available commercially (Thermo Fisher and Toronto Research), they are relatively new and don t have the publications or applications backing up their use as sulfo-SBED. A protocol for the use of these compounds in the study of protein interactions can be found in Chapter 28, Section 3.2. 

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. 

Sulfo-SBED is soluble in organic solvents, such as DMSO (125mM), DMF (170mM), and methanol (12mM), or to a lesser degree in pure water ( 5 mM). The solubility of Sulfo-SBED in... 

Figure 28.11 Sulfo-SBED is a label transfer agent that contains a water-soluble sulfo-NHS ester to label bait proteins and a phenyl azide group for photoreactive capture of a prey protein. The biotin label can be used for detection or isolation of protein-protein conjugates using (strept)avidin reagents. The stars indicate the atoms that were used to measure the indicated molecular dimensions.

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. 

A derivative of Sulfo-SBED containing a thiol-reactive pyridyl disulfide group on its thermo-reactive arm has been reported for modification of bait proteins containing a cysteine residue. 

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.

Figure 28.13 A sulfo-SBED-captured protein interaction can be released using DTT to cleave the disulfide within the cross-bridge leading to the bait protein. The result transfers the biotin label to the unknown interacting protein. The biotin tag thus allows the interacting protein to be detected or isolated using (strept)avidin reagents.

The following protocol represents a suggested method that will work well for many proteins. It is a blend of protocols used in the literature and recommended by Thermo Fisher in the Sulfo-SBED instruction manual. Modifications to reaction conditions may be necessary in certain cases to maintain protein stability or solubility, depending on the properties of the particular bait protein being used. 

Prepare a solution of Sulfo-SBED in dry DMF or DMSO at a concentration of 40 pg/ml. Protect from light. 

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. 

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