NHS ester labeling of amino biomolecules

NHS (N-HydroxySuccinimide) esters and other activated esters (sulfo-NHS, sulfotetrafluorophenyl - STP) are reactive compounds suitable for the modification of amino groups. NHS is most common type of activated esters.

Usual modifications are fluorescent labels, fluorescence quenchers, and other reporter groups. Alkyne and azido group can be attached using activated esters to adapt biomolecules to Click Chemistry.

Since amino groups are nearly always contained in proteins and peptides, modification of these biopolymers is especially common. Other examples are amino-oligonucleotides, amino-modified DNA, and amino-containing sugars.

The reaction of NHS esters with amines is strongly pH-dependent: at low pH, the amino group is protonated, and no modification takes place. At higher-than-optimal pH, hydrolysis of NHS ester is quick, and modification yield diminishes. Optimal pH value for modification is 8.3-8.5.

Water is most common solvent for the labeling. If NHS ester is poorly soluble, it can be added as a solution in DMSO or DMF to a solution of protein in water, adjusted to pH 8.3-8.5. Note that DMF must not contain amines (and thus should have no odor).

We recommend using the following general protocol for the labeling of biomolecules with NHS esters produced by Lumiprobe.

  1. Calculate required amount of NHS ester:

    NHS_ester_weight [mg] = 8 × amino_compound_weight [mg] × NHS_ester_molar_weight [Da] / amino_compound_molar_weight [Da].

    8 is molar excess of NHS ester. It is experimental value for mono-labeling, suitable for many common proteins and peptides. However, in some cases using less or more NHS ester is required. It depends on protein structure, reagent, and solubility. Molar weight of Lumiprobe products can be found on corresponding product pages.

    For example, to label 3 mg of BSA (molar weight 69300 Dalton) with Cy5 NHS ester (molar weight 616 Dalton), and obtain maximum yield of mono-labeled product, one should use
    8 × 3 mg × 616 Da / 69300 Da = 0.21 mg
    of Cy5 dye NHS ester.

  2. Determine volume of reaction mixture. The labeling can be performed on any scale from nanomols to dozens of grams. When the scale is low, use minimal volume (10-20 uL). Higher concentrations (1-10 mg of amino-biomolecule per mL of mixture) are optimal.

  3. Dissolve NHS ester in 1/10 reaction volume of DMF or DMSO. Amine-free DMF is preferred solvent. After the reaction, NHS ester can be stored in solution for 1-2 months at -20ºC.

  4. Dissolve biomolecule in 9/10 reaction volume of buffer with pH 8.3-8.5.

    0.1 M Sodium bicarbonate solution has appropriate pH. Another alternative is 0.1 M phosphate buffer. Note pH is the most important thing. Avoid using buffers containing amines (Tris can sometimes be used but not recommended).

    When doing large-scale labeling (hundreds of milligrams of NHS ester), note that the mixture tends to acidify with time because of hydrolysis of NHS ester. Monitor pH, or use more concentrated buffer then.

  5. Add NHS ester solution to the solution of biomolecule, and vortex well. Keep on ice overnight, or at room temperature during at least 4 hours.

  6. Purify the conjugate using appropriate method: gel-filtration for macromolecules is most universal. Precipitation and chromatography is another alternative. Organic impurities (such as N-hydroxysuccinimide, NHS ester, acid produced by hydrolysis) are almost always easily separated. For proteins and nucleic acids, ethanol or acetone precipitation can be used.

Related products

AF488 NHS ester

AF488 (Alexa Fluor 488) NHS ester, a bright, photostable and hydrophilic fluorescent dye for green channel.
Show pricing
Cat. # Quantity Price Lead time
11820 1 mg $110.00 in stock
21820 5 mg $290.00 in stock
41820 25 mg $690.00 in stock
51820 50 mg $1270.00 5 days
61820 100 mg $1990.00 in stock

Alkyne NHS ester (hexynoic acid NHS ester)

Alkyne NHS ester for the labeling of biomolecules with alkyne group for Copper catalyzed Click chemistry.
Show pricing
Cat. # Quantity Price Lead time
41720 25 mg $95.00 in stock
51720 50 mg $180.00 in stock
61720 100 mg $290.00 in stock
91720 1 g please inquire in stock

Azidobutyric acid NHS ester

Activated ester for azido labeling of peptides and proteins.
Show pricing
Cat. # Quantity Price Lead time
53720 50 mg $60.00 in stock
63720 100 mg $95.00 in stock
73720 1 g please inquire in stock
83720 5 g please inquire in stock

BDP 558/568 NHS ester

BDP 558/568 is a fluorophore for the yellow region of the spectrum. Like other BDP dyes, this fluorophore has high fluorescence quantum yield, brightness, and photostability.
Show pricing
Cat. # Quantity Price Lead time
17420 1 mg $110.00 in stock
27420 5 mg $210.00 in stock
47420 25 mg $410.00 in stock
57420 50 mg $695.00 in stock
67420 100 mg $1190.00 in stock

BDP 650/665 X NHS ester

Amine reactive of BDP 650/665, a borondipyrromethene dye for Cy5 channel, possessing a high molar extinction coefficient, and a good emission quantum yield.
Show pricing
Cat. # Quantity Price Lead time
1E420 1 mg $110.00 in stock
2E420 5 mg $210.00 in stock
4E420 25 mg $410.00 in stock
5E420 50 mg $695.00 in stock
6E420 100 mg $1190.00 in stock

BDP FL NHS ester

Amino-reactive BDP FL, a very bright and photostable dye for fluorescein channel.
Show pricing
Cat. # Quantity Price Lead time
11420 1 mg $110.00 in stock
21420 5 mg $210.00 in stock
41420 25 mg $410.00 in stock
51420 50 mg $695.00 in stock
61420 100 mg $1190.00 in stock

BDP R6G NHS ester

BDP R6G is a borondipyrromethene dye which has absorption and emission spectra similar to R6G.
Show pricing
Cat. # Quantity Price Lead time
14420 1 mg $110.00 in stock
24420 5 mg $210.00 in stock
44420 25 mg $410.00 in stock
54420 50 mg $695.00 in stock
64420 100 mg $1190.00 in stock

BDP TR X NHS ester

BDP TR X NHS ester is a derivative of BDP TR containing a long linker based on aminohexanoic acid (C6). The dye has absorption and emission close to ROX (Texas Red). The NHS ester function can be used for the conjugation with proteins and peptides.
Show pricing
Cat. # Quantity Price Lead time
1D420 1 mg $110.00 in stock
2D420 5 mg $210.00 in stock
4D420 25 mg $410.00 in stock
5D420 50 mg $695.00 in stock
6D420 100 mg $1190.00 in stock

Cyanine3 NHS ester

Amine-reactive Cyanine3 dye NHS ester.
Show pricing
Cat. # Quantity Price Lead time
11020 1 mg $110.00 in stock
21020 5 mg $210.00 in stock
41020 25 mg $410.00 in stock
51020 50 mg $695.00 in stock
61020 100 mg $1190.00 in stock

Cyanine3.5 NHS ester

Cyanine3.5 dye NHS ester derivative.
Show pricing
Cat. # Quantity Price Lead time
12020 1 mg $110.00 in stock
22020 5 mg $210.00 in stock
42020 25 mg $410.00 in stock
52020 50 mg $695.00 in stock
62020 100 mg $1190.00 in stock

Cyanine5 NHS ester

Amine-reactive Cyanine5 activated ester for the labeling of proteins, peptides, and other molecules.
Show pricing
Cat. # Quantity Price Lead time
13020 1 mg $110.00 in stock
23020 5 mg $210.00 in stock
43020 25 mg $410.00 in stock
53020 50 mg $695.00 in stock
63020 100 mg $1190.00 in stock

Cyanine5.5 NHS ester

Cyanine5.5 NHS is far-red / near-infrared amine-reactive dye.
Show pricing
Cat. # Quantity Price Lead time
17020 1 mg $110.00 in stock
27020 5 mg $210.00 in stock
47020 25 mg $410.00 in stock
57020 50 mg $695.00 in stock
67020 100 mg $1190.00 in stock

Cyanine7 NHS ester

Amine reactive NHS ester of near infrared dye Cyanine7.
Show pricing
Cat. # Quantity Price Lead time
15020 1 mg $110.00 in stock
25020 5 mg $210.00 in stock
45020 25 mg $410.00 in stock
55020 50 mg $695.00 in stock
65020 100 mg $1190.00 in stock

Cyanine7.5 NHS ester

Amine reactive NHS ester of NIR fluorescent dye Cyanine7.5.
Show pricing
Cat. # Quantity Price Lead time
16020 1 mg $110.00 in stock
26020 5 mg $210.00 in stock
46020 25 mg $410.00 in stock
56020 50 mg $695.00 in stock
66020 100 mg $1190.00 in stock

FAM NHS ester, 6-isomer

Activated N-hydroxysuccinimide ester of fluorescein (FAM), pure 6-isomer.
Show pricing
Cat. # Quantity Price Lead time
15120 1 mg $110.00 in stock
25120 5 mg $160.00 in stock
45120 25 mg $210.00 in stock
55120 50 mg $270.00 in stock
65120 100 mg $420.00 in stock

Pentynoic acid STP ester

An activated ester for the introduction of alkyne into peptides/proteins.
Show pricing
Cat. # Quantity Price Lead time
30720 10 mg $110.00 21 days
40720 25 mg $210.00 in stock
50720 50 mg $410.00 in stock
60720 100 mg $695.00 5 days

Pyrenebutyric acid NHS ester

Pyrene NHS ester for the labeling of biomolecules with pyrene residue. Pyrene is a fluorescent proximity probe which forms excimers, and it is also an anchor group for graphite immobilization.
Show pricing
Cat. # Quantity Price Lead time
63520 100 mg $110.00 in stock

Sulfo-Cyanine3 NHS ester

Water soluble sulfo-Cyanine3 SE activated ester for amino-biomolecule labeling.
Show pricing
Cat. # Quantity Price Lead time
11320 1 mg $110.00 in stock
21320 5 mg $290.00 in stock
41320 25 mg $690.00 in stock
51320 50 mg $1270.00 in stock
61320 100 mg $1990.00 in stock

Sulfo-Cyanine5 NHS ester

Water-soluble Cyanine 5 succinimide ester.
Show pricing
Cat. # Quantity Price Lead time
13320 1 mg $110.00 in stock
23320 5 mg $290.00 in stock
43320 25 mg $690.00 in stock
53320 50 mg $1270.00 in stock
63320 100 mg $1990.00 in stock

Sulfo-Cyanine5.5 NHS ester

Water soluble, sulfonated, far-red sulfo-Cyanine5.5 dye in the form of NHS ester.
Show pricing
Cat. # Quantity Price Lead time
17320 1 mg $110.00 in stock
27320 5 mg $290.00 in stock
47320 25 mg $690.00 in stock
57320 50 mg $1190.00 in stock
67320 100 mg $1850.00 in stock

Sulfo-Cyanine7 bis-NHS ester

Sulfo-Cyanine7 bis-NHS ester is a bifunctional derivative of water soluble near infrared sulfo-Cyanine7. It contains two activated ester functional groups.
Show pricing
Cat. # Quantity Price Lead time
153D0 1 mg $110.00 in stock
253D0 5 mg $290.00 in stock
453D0 25 mg $690.00 in stock
553D0 50 mg $1270.00 in stock
653D0 100 mg $1990.00 in stock

Sulfo-Cyanine7 NHS ester

Sulfo-Cyanine7 NHS ester is a water soluble, near infrared, amine-reactive dye.
Show pricing
Cat. # Quantity Price Lead time
15320 1 mg $110.00 in stock
25320 5 mg $290.00 in stock
45320 25 mg $690.00 in stock
55320 50 mg $1270.00 in stock
65320 100 mg $1990.00 in stock

TAMRA NHS ester, 5-isomer

Amine-reactive, activated N-hydroxysuccinimidyl ester of TAMRA dye for the labeling of amine groups of proteins, peptides, and amino-oligonucleotides.
Show pricing
Cat. # Quantity Price Lead time
17120 1 mg $110.00 in stock
27120 5 mg $210.00 in stock
47120 25 mg $410.00 in stock
57120 50 mg $695.00 in stock
67120 100 mg $1190.00 in stock

Your item has been added. View your cart or proceed to checkout
The count of items is incorrect.