Thermo Scientific EZ-Link NHS-Iminobiotin is an amine-specific biotinylation reagent that uses a modified form of biotin to enable soft-release, reversible binding to streptavidin in affinity purification methods.
NHS-Iminobiotin is a version of NHS-Biotin in which the terminal oxygen atom in the biotin ring-structure is replaced with nitrogen. This modification slightly weakens the otherwise very strong binding interaction between biotin and avidin (or streptavidin); therefore, NHS-Iminobiotin can be used for affinity purification methods that require soft-release, reversible binding. (The molecule is supplied as a trifluoroacetamide.) Like other NHS-ester reagents, this compound is for labeling amine-containing molecules such as antibodies or other proteins.
- Reversible binding to avidin prevents protein denaturation during purification
- Binds to avidin at pH greater than 9.5 and dissociates at pH 4.0
- Reacts with primary amines at pH 7 to 9 to form a stable amide bond
||Chemical structure of NHS-Iminobiotin. This biotinylation reagent labels antibodies and other proteins and molecules that contain primary amines (e.g., side-chain of lysine). The trifluoroacetamide (TFA) group dissociates from the rest of the molecule when dissolved. For more information, see our review of NHS Ester Reaction Chemistry.
Properties of NHS-Iminobiotin.
||Iminobiotin NHS ester
||C16H19F3N4O5S or C16H19F3N4O5S
||436.21 (or 340.40 without TFA)
|Spacer arm length
|Mass added to target
||256.14 daltons (assuming no TFA in final product)
||White to off-white solid
||At least 10mg/mL in water
||4°C with desiccant, protect from moisture, use only fresh solutions
||N-hydroxysuccinimide ester, reacts with primary amines at pH 7.0-9.0
We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.
N-Hydroxysulfosuccinimide (NHS) esters of biotin are 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.
Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.
Biotinylation Reagents Selection Guide
Review of Biotinylation Methods and Applications
Chemistry of Crosslinking (and Labeling Reagents)
All Amine-Reactive Biotinylation Reagents
All Biotinylation Reagents
Biotin Quantitation Reagents and Kits
Avidin, Streptavidin, NeutrAvidin Affinity Resins