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Amine-reactive biotinyltation reagent
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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.


  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..


• Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
• Intracellular labeling—membrane-permeable, can be used to label inside cells
• Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the N-terminal-amine
• Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
• Solubility—water insoluble, must be dissolved in DMSO or DMF before further dilution in aqueous buffers
• Very short—spacer arm is 13.5 angstroms; it consists of the native biotin valeric acid group only

Product Citation

Chemical Properties

Physical AppearanceA solid
StorageDesiccate at -20°C
Cas No.35013-72-0
SynonymsBiotin N-hydroxysuccinimide ester, Biotin-OSU, Biotin NHS, Biotin SE
Solubility≥17.069mg/mL in DMSO, <2.63 mg/mL in EtOH, <2.67 mg/mL in H2O
Chemical Name(2,5-dioxopyrrolidin-1-yl) 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate
SDFDownload SDF
Shipping ConditionEvaluation sample solution : ship with blue ice.All other available size: ship with RT , or blue ice upon request
General tipsFor obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.


Biotinylation method [1]:


whole blood

Preparation method

Soluble in DMSO or DMF.

Reaction Conditions

30 minutes


Firstly, NHS-biotin was dissolved in DMSO at a concentration of 100 mg/mL, after that using 90% DMSO solution diluted the biotin to a concentration of 20 mg/mL. The biotin solution was sterilized by filtering the solution through 0.2-μm pore nylon syringe filters and the solution was diluted with 6 volumes 0.9% saline solution. Blood was allowed to warm to room temperature prior to labeling with NHS-biotin. To achieve a biotin concentration of 0.04 pg of biotin/RBC(Red Blood Cells), Biotin-DMSO-saline solution was injected into each blood bag and agitated for 30 minutes.


[1]. J.E. Tomlinson, E. Taberner, R.C. Boston, S.D. Owens, and R.D. Nolen-Walston. Survival Time of Cross-Match Incompatible Red Blood Cells in Adult Horses. J Vet Intern Med 2015;29:1683–1688.

Biological Activity

Description NHS-Biotin is the simplest NHS-ester activated biotinylation reagent used for intracellular labeling.
Targets intracellular proteins          

Quality Control

Chemical structure


Related Biological Data


Related Biological Data


Related Biological Data


Related Biological Data


Related Biological Data