Biotin-dPEG®₁₂-TFP ester

Product number 10008, Biotin-dPEG®12-TFP ester, is the 2,3,5,6-tetrafluorophenyl (TFP) ester analog of PN10198, NHS-dPEG®12-biotin. PN10008 is part of a family of dPEG®-biotin reagents that provide different lengths of dPEG® between the biotin moiety and the reactive end group. The active esters can be either N-hydroxysuccinimide (NHS) or 2,3,5,6-tetrafluorophenol (TFP). Compared to NHS esters, TFP esters are more reactive towards primary amines and more stable in water or aqueous buffer. The optimum pH for reaction with TFP esters is in the range of 7.5 – 8.5, and for NHS esters the optimum reaction pH range is 6.0 – 7.5.

Biotinylation is the process of covalently attaching biotin to a molecule such as a peptide, protein, or nucleic acid, or to a surface such as glass or gold. It is widely used in bioconjugation research and product development. Biotin is normally poorly soluble in water, but the amphiphilic dPEG® linker imparts excellent solubility in water or aqueous buffer and in organic solvent. The 2,3,5,6-tetrafluorophenyl (TFP) moiety of biotin-dPEG®12-TFP ester reacts with free primary amines, such as the amines on the side chain of lysine, forming a stable amide bond. The biotin moiety is used in labeling experiments, supramolecular construction, affinity chromatography, the creation of biotinylated antibodies, enzyme-linked immunosorbent assays (ELISA), pull-down assays, and many other applications.

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. Unfortunately, both reagents are highly problematic for biochemical applications. Insoluble in water, these reagents must be dissolved in organic solvent immediately prior to 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.

Biotin-dPEG®12-TFP ester does not cause protein aggregation. This permits experiments that require a high degree of biotin labeling without concerns about loss of protein due to aggregation. Dissolution of the product in organic solvent is not required for conjugation, thus providing easier reaction setup and greater reaction reproducibility. The dPEG® linker in PN10008 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®12-biotin provides better access of the biotin to the avidin or streptavidin binding pocket compared to these older, hydrophobic reagents.

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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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