NHS-iminobiotin

The iminobiotin—avidin interaction also can be utilized in the opposite approach. Immobilized iminobiotin affinity columns can be used to purify avidin- or strep-tavidin-containing complexes under mild elution conditions (Hofmann et al., 1980). 

NHS-iminobiotin is insoluble in aqueous solution. It can be dissolved in organic solvent (DMF) prior to addition of a small aliquot to a buffered reaction medium. Do not exceed 10% DMF in the reaction, to avoid protein precipitation problems. Optimal conditions for protein derivatization include non-amine-containing buffers at a pH of 7—9. The following protocol is a suggested method for labeling antibodies with NHS-iminobiotin. Some optimization may have to be done for particular derivatization needs. 

Add 100 xl of the NHS-iminobiotin solution to each milliliter of the antibody solution. Mix well to dissolve. Note some turbidity may be present in the reaction due to incomplete dissolution of the NHS-iminobiotin. The solution may look cloudy or have a microparticulate suspension present. This is normal for many water-insoluble reagents when added to an aqueous solution in an organic solvent. As the reaction takes place, the NHS-iminobiotin will be driven into solution, both by coupling to the protein and by hydrolysis of the NHS ester. 

Remove unreacted NHS-iminobiotin and reaction by-products by dialysis or gel filtration using a column of Sephadex G-25. 

NHS-iminobiotin can be used to label amine-containing molecules, creating amide linkages. 

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NHS-iminobiotin can be used to label amine-containing molecules with an iminobiotin tag, providing reversible-binding potential with avidin or streptavidin. The NHS ester reacts with proteins and other amine-containing molecules to create stable amide bond derivatives (Figure 11.6). An iminobiotinylated molecule then can be used to target and purify other... 

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]. 

The application of biotinylated receptor substrates is a useful approach, incubating the biotinylated substrates with their specific receptors prior to isolation on an avidin-coated support.In such cases, biotinylation with a cleavable reagent such as Sulfo-NHS-SS-biotin or NHS-Iminobiotin is essential for recovery of the isolated receptor. Alternatively, the receptor can be recovered by substrate competition. 

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