NHS-Biotin

NHS-Biotin (C₁₄H₁₈O₅N₃S) is N-hydroxysuccinimido biotin. It is the shortest of three similar EZ-Link NHS-Biotin Reagents that enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing biomolecules in solution. NHS-Biotin offers researchers the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor. Because it is uncharged and contain simple alkyl-chain spacer arms, NHS-Biotin compound is membrane-permeable and useful for intracellular labeling.

NHS-Biotin is the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

 Ref:

1. Fouassier, L., Yun, C. C., Fitz, J. G. and Doctor, R. B. (2000). Evidence for Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) Self-association through PDZ-PDZ Interactions. J. Biol. Chem. 275, 25039-25045.
2. Nunomura, W., Takakuwa, Y., Parra, M., Conboy, J.G., & Mohandas, N. (2000) Regulation of protein 4.1R, p55 and glycophorin C ternary complex in human erythrocyte membrane. J. Biol. Chem. 275(32): 24540-24546.
3. Chiu, N.H. & T.K. Christopoulos. 1999. Two-site expression immunoassay using a firefly luciferase-coding DNA label. Clin. Chem. 45: 1954–1959..

Assessment of solvent residues accessibility using three Sulfo-NHS-biotin reagents in parallel: application to footprint changes of a methyltransferase upon binding its substrate.[Pubmed:17968972]

J Mass Spectrom. 2008 Mar;43(3):360-70.

NHS-Biotin modification as a specific lysine probe coupled to mass spectrometry detection is increasingly used over the past years for assessing amino acid accessibility of proteins or complexes as an alternative when well-established methods are challenged. We present a strategy based on usage in parallel of three commercially available reagents (Sulfo-NHS-Biotin, Sulfo-NHS-LC-biotin, and Sulfo-NHS-LC-LC-biotin) to efficiently assess the solvent accessibility of amino acids using MALDI-TOF mass spectrometry. The same qualitative pattern of reactivity was observed for these three reagents on the THUMPalpha protein at four reagent/polypeptide molar ratios (2 : 1, 6 : 1, 13 : 1, and 26 : 1). Peptide assignment of the detected ions gains in accuracy because of the triple redundancy due to specific increments of monoisotopic mass. These reagents are a good alternative to isotope labeling when using only a single MALDI-TOF mass spectrometer. We observed that hydroxyl groups of serine and tyrosine residues were also modified by these Sulfo-NHS-Biotin reagents. The low amount of protein required and the method's simplicity make this procedure accessible and affordable in order to obtain topological information on proteins difficult to purify. This method was used to identify two lysine residues of the TrmG10 methyltransferase from Pyrococcus abyssi that were differentially reactive, modified in the protein but not in the tRNA-protein complex.

The beta chain of the GPIIb molecule on ruminant leukocytes and platelets is not labelled by the sulfo-NHS-biotin method.[Pubmed:8578739]

Vox Sang. 1995;69(3):248-9.

Glycoprotein (Gp)IIb/IIIa molecules have been immunoprecipitated from platelets and leukocytes of cattle, goat, horse, human, sheep and swine using specific monoclonal antibodies. The sulfo-NHS-Biotin (sulfosuccinimidobiotin) method used to label the proteins has been found unsuitable for labelling the beta chain of the ruminant GPIIb/IIIa molecule. The beta chain was present on ruminant leukocytes and platelets when immunoprecipitates were silver stained.