Sulfo-NHS-SS-biotin

Sulfo-NHS-SS-Biotin SuIfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate MW 606.7 24.3 A 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents 

Dissolve the antibody to be biotinylated in 50 mM sodium bicarbonate, pH 8.5, at a concentration of 10 mg/ml. Other buffers and pH conditions between pH 7 and 9 can be used as long as no amine-containing buffers like Tris are present. Avoid also the presence of disulfide reducing agents that can cleave the disulfide group of the biotinylation reagent. 

3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

Remove unreacted biotinylation reagent and reaction by-products by dialysis or gel filtration using a desalting resin. 

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Figure 11.7 Sulfo-NHS-SS-biotin reacts with amine groups to form amide bonds. The biotin group can be later cleaved off the modified molecule by reduction of its internal disulfide linkage.

Dissolve 12 mg of sulfo-NHS-SS-biotin in 48 ml of cold PBS and immediately add 10 ml of the solution to each flask containing the washed cells. 

The application of biotinylated receptor substrates is another approach, incubating the labeled substrate with the receptors prior to isolation on an avidin-coated support. In such cases, biotinylation with a cleavable biotinylation reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin would be essential for recovery of the isolated receptor. Alternatively, the receptor could be recovered by substrate competition. Perhaps one of the major drawbacks to the application of affinity techniques is the relative low molecular weight or small size of the receptor substrates, making them difficult ligands to immobilize. However, affinity procedures have been applied to the purification of a number of different receptors although relatively little work has been reported on those involved in the processing of neurotransmittors, neuropeptides, and hormones [1,2]. 

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