NHS-dPEG®₂₄-biotin

NHS-dPEG®24-biotin, product number 10774, is an extended version of our consistently popular biotinylation products. Numerous publications feature the use of this product. Although biotin usually is poorly soluble in water, the amphiphilic dPEG® linker creates excellent water solubility while maintaining organic solvent solubility. The N-hydroxysuccinimidyl (NHS) moiety of NHS-dPEG®24-biotin reacts with free primary amines, such as the amines on the side chain of lysine, forming a stable amide bond. Labeling experiments, supramolecular construction, affinity chromatography, pull-down assays, the creation of biotinylated antibodies, and many other applications use biotin to take advantage of the exceptionally high affinity between biotin and avidin or streptavidin.

Conventional Biotinylation Reagents Compared to NHS-dPEG®24-biotin

NHS-LC-biotin (biotin coupled to aminocaproic acid and activated with N-hydroxysuccinimide) and NHS-LC-LC-biotin (with two aminocaproic acid chains) are widely used biotinylation reagents. Both reagents, though, are highly problematic for biochemical applications. Insoluble in water, both reagents must be dissolved in organic solvent immediately before use. When coupled to proteins, the linkers' high degree of hydrophobicity causes rapid aggregation and deactivation of the proteins. A variant of NHS-LC-biotin, known as Sulfo-NHS-LC-biotin, is water-soluble, but it still causes rapid aggregation of proteins.

NHS-dPEG®24-biotin does not cause protein aggregation. Consequently, customers requiring a high degree of biotin labeling can obtain such labeling without concerns about protein loss due to aggregation. Dissolution of the product in an organic solvent is not required for conjugation, thus providing simpler reaction setup and higher reaction reproducibility. The dPEG® linker in PN10774 is significantly longer than the aminocaproic acid linker in LC-biotin and longer than the two aminocaproic acid linkers in LC-LC-biotin. Consequently, dPEG®24-biotin provides better access of the biotin to the avidin or streptavidin binding pocket compared to these older, hydrophobic reagents.

Additional Biotinylation Reagents Besides NHS-dPEG®24-biotin
NHS-dPEG®24-biotin is part of a family of dPEG®-biotin reagents that provide different lengths. The active esters can be either NHS or 2,3,5,6-tetrafluorophenol (TFP). Compared to NHS ester, TFP esters are more reactive towards primary amines and more stable in water or aqueous buffer. The optimum pH for reaction with NHS esters is in the range of 7.2 – 7.5, while for TFP esters, the optimum reaction pH range is slightly higher.

Application References:
Hermanson, G. T. Chapter 11, (Strept)avidin-Biotin Systems. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 465-505.
Hermanson, G. T. Chapter 18, PEGylation and Synthetic Polymer Modification. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 787-838.

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