NHS Ester Labeling of Biomolecules Containing Primary Amino Groups
NHS (N-HydroxySuccinimide) esters and other activated esters (sulfo-NHS, sulfotetrafluorophenyl — STP) are reactive compounds suitable for the modification of amino groups. NHS is the most common type of activated esters.
Usual modifications are fluorescent labels, fluorescence quenchers, and other reporter groups. Alkyne and azido groups can be attached using activated esters to adapt biomolecules to click chemistry.
Especially common are modifications of proteins and peptides since they nearly always contain amino groups. Other examples are modifications of amino-oligonucleotides, amino-modified DNA, and amino-containing sugars.
The reaction of NHS esters with amino groups is strongly pH-dependent: at low pH, the amino group is protonated, and the modification does not occur. At higher-than-optimal pH, hydrolysis of NHS ester is quick, and the yield of modified molecule decreases. The optimal pH value for modification is 8.3-8.5.
Water is the most common solvent used to dissolve NHS esters for biomolecule labeling. If NHS ester is poorly soluble in aqueous solutions, it can be at first dissolved in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and then added to a solution of protein in a buffer with pH 8.3-8.5. Note that DMF can degrade into dimethylamine, which has a fishy smell and is able to react with activated esters. Therefore, to label biomolecules, it is important to use high-quality DMF that contains no dimethylamine and has no fishy odor.
We recommend using the following general protocol for the labeling of biomolecules with NHS esters produced by Lumiprobe:
- Calculate the required amount of NHS ester using the formula below or our protein labeling calculator: NHS_ester_weight [mg] = 8 × amino_compound_weight [mg] × NHS_ester_molar_weight [Da] / amino_compound_molar_weight [Da]. 8 is a molar excess of NHS ester. It is an empirical value for mono-labeling, suitable for many common proteins and peptides. However, in some cases using less or more NHS ester can increase the yield of the modified biomolecule. It depends on protein structure, reagent used for labeling, and its solubility. The molar weight of Lumiprobe products can be found on the corresponding product pages. For example, to label 3 mg of BSA (molar weight 66500 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 / 66500 Da = 0.22 mg of Cy5 dye NHS ester.
- Calculate the volume of the reaction mixture. The optimal biomolecule concentration in labeling reaction is 1-10 mg per mL. The labeling can be performed on any scale, from nanomols to dozens of grams. If you label a low amount of biomolecule, keep the volume of the reaction mixture at a minimum (10-20 uL).
- Dissolve NHS ester in 1/10 reaction volume of water, DMF, or DMSO. Dimethylamine-free DMF or water are preferred solvents. NHS ester dissolved in DMF can be stored in solution for 1-2 months at −20ºC. The aqueous solution of NHS ester should be used immediately after preparation.
- 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 that pH is the most important factor for successfully labeling biomolecule with NHS ester. Avoid buffers containing amines (Tris-based buffers can sometimes be used but are not recommended. Tris contains amino group, but its affinity to activated esters is low). When doing large-scale labeling (using hundreds of milligrams of NHS ester), note that the mixture tends to acidify with time because of the hydrolysis of NHS ester. Monitor pH, or use a more concentrated buffer then.
- Add NHS ester solution to the solution of biomolecule, and vortex well. Keep on ice overnight, or at room temperature from 1 to 4 hours.
- Purify the conjugate using the appropriate method: gel-filtration for macromolecules is the most common. Chromatography is another alternative. For proteins and nucleic acids, ethanol or acetone precipitation can be used. Organic impurities (such as N-hydroxysuccinimide, NHS ester, and acid produced by hydrolysis) are almost always easily separated by these methods.
Related products
3-Maleimidopropionic acid NHS-ester
Bifunctional crosslinker with an amine-reactive succinimidyl ester and a thiol-reactive maleimide group.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 3419-100mg | 100 mg |
–
|
in stock | |
| 3419-500mg | 500 mg |
$110
|
in stock |
AF 488 TFP ester
Tetrafluorophenyl ester of green fluorescent dye AF 488 for labeling amino groups in proteins, peptides, and modified oligonucleotides.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 118T0 | 1 mg |
$165
|
in stock | |
| 218T0 | 5 mg |
$510
|
in stock | |
| 418T0 | 25 mg |
$1650
|
in stock |
Alkyne-PEG2-NHS ester
Bifunctional hydrophilic linker with NHS ester and terminal alkyne functional group based on diethyleneglycol (PEG2).
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2219-1g | 1 g |
$450
|
in stock | |
| 2219-5g | 5 g |
$980
|
in stock |
Alkyne-PEG3-NHS ester
Bifunctional hydrophilic linker with NHS ester and terminal alkyne functional group based on triethyleneglycol (PEG3).
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2298-1g | 1 g |
$490
|
in stock | |
| 2298-5g | 5 g |
$980
|
in stock |
Alkyne-PEG4-NHS ester
Bifunctional hydrophilic linker with NHS ester and terminal alkyne functional group based on tetraethyleneglycol (PEG4).
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2409-100mg | 100 mg |
$250
|
in stock | |
| 2409-1g | 1 g |
$790
|
in stock |
ATFB SE (N-Succinimidyl 4-Azidotetrafluorobenzoate)
N-Succinimidyl 4-Azidotetrafluorobenzoate (ATFB SE, ATFB-OSu) is a photo-activatable linker used as a versatile labeling agent for investigating biological receptors.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2574-250mg | 250 mg |
$134
|
in stock | |
| 2574-1g | 1 g |
$419
|
in stock |
ATT 550 NHS ester
ATT 550 succinimidyl ester for labeling various amine-containing molecules in the aqueous phase.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 1C920 | 1 mg |
$159
|
in stock | |
| 2C920 | 5 mg |
$640
|
in stock | |
| 4C920 | 25 mg |
$1640
|
in stock | |
| 5C920 | 50 mg |
$2090
|
in stock | |
| 6C920 | 100 mg |
$2840
|
in stock |
ATT 647N NHS ester
ATT 647N succinimidyl ester for labeling various amine-containing molecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 1E920 | 1 mg |
$199
|
in stock | |
| 2E920 | 5 mg |
$746
|
in stock |
Azide-PEG6-NHS ester
A hydrophilic bifunctional linker with azide and NHS ester function groups flanking PEG6 (hexaethylene glycol).
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 3692-250mg | 250 mg |
$325
|
in stock | |
| 3692-1g | 1 g |
$539
|
in stock |
Azidobutyric acid NHS ester
Activated NHS ester for azido-labeling of peptides and proteins.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 53720 | 50 mg |
$110
|
in stock | |
| 63720 | 100 mg |
$210
|
in stock | |
| 73720 | 1 g |
$950
|
in stock | |
| 83720 | 5 g |
please inquire
|
in stock |
BDP 564/570 NHS ester
Lypohilic borodipyrromethene dye with orange emission for the labeling of primary and secondary amine groups.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 1H420 | 1 mg |
$125
|
in stock | |
| 2H420 | 5 mg |
$325
|
in stock | |
| 4H420 | 25 mg |
$510
|
in stock | |
| 5H420 | 50 mg |
$895
|
in stock | |
| 6H420 | 100 mg |
$1490
|
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 | Buy this product |
|---|---|---|---|---|
| 14420 | 1 mg |
$125
|
in stock | |
| 24420 | 5 mg |
$260
|
in stock | |
| 44420 | 25 mg |
$510
|
in stock | |
| 54420 | 50 mg |
$895
|
in stock | |
| 64420 | 100 mg |
$1490
|
in stock |
BDP® TMR-X-NHS ester
Amine reactive NHS ester of BDP TMR, bright borondipyrromethene dye for TAMRA channel especially suitable for fluorescence polarization assays. This compound contains an aminohexanoic acid (C6) linker between the dye and the functional group.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 1F420 | 1 mg |
$125
|
in stock | |
| 2F420 | 5 mg |
$260
|
in stock | |
| 4F420 | 25 mg |
$510
|
in stock | |
| 5F420 | 50 mg |
$895
|
in stock | |
| 6F420 | 100 mg |
$1490
|
in stock |
Biotin-PEG4-NHS ester
PEG4-containing activated ester for simple and efficient biotin labeling of antibodies, proteins, and other primary amine-containing biomolecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2893-100mg | 100 mg |
$145
|
in stock | |
| 2893-1g | 1 g |
$590
|
in stock |
Biotin-XX-NHS ester
Biotin derivative with long spacer to reduce steric effect when binding amino acids, peptides, or proteins by reacting with primary amines.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2897-100mg | 100 mg |
$225
|
in stock | |
| 2897-1g | 1 g |
$670
|
in stock |
DBCO-NHS ester
Azodibenzocyclooctyne (DBCO, ADIBO) reagent for strain-promoted copper-free click chemistry (SPAAC). The reagent contains an NHS ester function for the attachment of the cyclooctyne to various molecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 34720 | 10 mg |
$80
|
in stock | |
| 44720 | 25 mg |
$110
|
in stock | |
| 54720 | 50 mg |
$135
|
in stock | |
| 64720 | 100 mg |
$210
|
in stock | |
| 94720 | 1 g |
$970
|
in stock | |
| K4720 | 5 g |
$3570
|
in stock |
DBCO-PEG6-NHS ester
A heterobifunctional linker with DBCO and NHS ester function groups flanking PEG6 (hexaethylene glycol).
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 3664-10mg | 10 mg |
–
|
in stock | |
| 3664-50mg | 50 mg |
$247
|
in stock | |
| 3664-100mg | 100 mg |
$449
|
in stock | |
| 3664-1g | 1 g |
$3490
|
5 days |
DBCO-STP ester
Activated ester for modification of proteins/antibodies/peptides followed by introduction into copper-free click chemistry reaction.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2584-5mg | 5 mg |
–
|
in stock | |
| 2584-10mg | 10 mg |
$90
|
in stock | |
| 2584-25mg | 25 mg |
$180
|
in stock | |
| 2584-100mg | 100 mg |
$450
|
in stock | |
| 2584-500mg | 500 mg |
$1700
|
in stock | |
| 2584-1g | 1 g |
$3000
|
in stock | |
| 2584-5g | 5 g |
please inquire
|
in stock |
Disuccinimidyl glutarate (DSG)
Homobifunctional crosslinker with two NHS ester groups at each end of a short 5-atom (7.7 angstrom) spacer.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 4018-100mg | 100 mg |
$95
|
in stock | |
| 4018-1g | 1 g |
$410
|
in stock |
DOTA-NHS ester
Bifunctional linker with DOTA chelating and amine-reactive NHS ester groups for bioconjugation.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 4245-100mg | 100 mg |
$125
|
in stock | |
| 4245-500mg | 500 mg |
$525
|
in stock | |
| 4245-1g | 1 g |
$990
|
in stock |
DOTA-tris-tBu mono-NHS ester
Bifunctional linker with DOTA chelating and amine-reactive NHS ester groups for bioconjugation.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2782-100mg | 100 mg |
$110
|
in stock | |
| 2782-500mg | 500 mg |
$485
|
in stock | |
| 2782-1g | 1 g |
$895
|
in stock |
DusQ® 1 NHS ester
Functionalized DusQ® 1 quencher derivative with quenching range from 480 - 580 nm for labeling of amine-containing molecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 21D20 | 5 mg |
–
|
in stock | |
| 41D20 | 25 mg |
$315
|
in stock | |
| 51D20 | 50 mg |
$580
|
in stock | |
| 61D20 | 100 mg |
$885
|
in stock |
DusQ® 21 NHS ester
Amino-reactive NHS ester with quencher for the conjugation with amines-containing molecules, DusQ® 21 quenching range 590 - 720 nm.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 28D20 | 5 mg |
–
|
in stock | |
| 48D20 | 25 mg |
$315
|
in stock | |
| 68D20 | 100 mg |
$885
|
in stock |
DusQ® 2 NHS ester
DusQ® 2 dark quencher with a quenching range from 560 - 670 nm with primary amine reactivity for synthesis of FRET probes. This NHS ester can be coupled to various molecules bearing amino groups.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 22D20 | 5 mg |
–
|
in stock | |
| 42D20 | 25 mg |
$315
|
in stock | |
| 52D20 | 50 mg |
$580
|
in stock | |
| 62D20 | 100 mg |
$885
|
in stock |
DusQ® 3 NHS ester
Amino-reactive NHS ester for the labeling with far-red quencher DusQ® 3.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 23D20 | 5 mg |
–
|
in stock | |
| 43D20 | 25 mg |
$315
|
in stock | |
| 53D20 | 50 mg |
$580
|
in stock | |
| 63D20 | 100 mg |
$885
|
in stock |
DusQ® 9 NHS ester
Functionalized DusQ® 9 quencher derivative with quenching range from 500 - 600 nm for labeling amine-containing molecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 47D20 | 25 mg |
$315
|
in stock | |
| 57D20 | 50 mg |
$580
|
in stock | |
| 67D20 | 100 mg |
$885
|
in stock |
Exo-BCN-PNP
BCN-PNP carbonate is a tool to introduce BCN moety via carbamate linkage with primary amino groups of substrate.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 4043-100mg | 100 mg |
$280
|
in stock | |
| 4043-500mg | 500 mg |
$990
|
in stock |
HEX NHS ester, 6-isomer
NHS-ester of HEX dye for proteins, peptides, and modified oligonucleotides labeling via reaction with amino groups.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 2B220 | 5 mg |
$175
|
in stock |
Hexynoic acid NHS ester
Hexynoic acid NHS ester for the labeling of biomolecules with alkyne group for copper-catalyzed click chemistry.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 41720 | 25 mg |
$95
|
in stock | |
| 51720 | 50 mg |
$180
|
in stock | |
| 61720 | 100 mg |
$290
|
in stock | |
| 91720 | 1 g |
please inquire
|
in stock |
Hexynoic acid STP ester
Alkyne activated ester for the modification of amine groups. STP ester group is an excellent alternative to NHS ester functionality.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 569-25mg | 25 mg |
$95
|
in stock | |
| 569-100mg | 100 mg |
$185
|
in stock | |
| 569-500mg | 500 mg |
$850
|
in stock |
JOE NHS ester, 5-isomer
JOE is a fluorescent dye with emission in the yellow part of the spectrum. This derivative is an activated NHS ester for conjugation with amines.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 11120 | 1 mg |
$110
|
in stock | |
| 21120 | 5 mg |
$210
|
in stock | |
| 41120 | 25 mg |
$410
|
in stock | |
| 51120 | 50 mg |
$695
|
in stock | |
| 61120 | 100 mg |
$1190
|
in stock |
Pentynoic acid STP ester
An activated ester for the introduction of alkyne into peptides/proteins and click reaction.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 30720 | 10 mg |
$110
|
in stock | |
| 40720 | 25 mg |
$210
|
in stock | |
| 50720 | 50 mg |
$410
|
in stock | |
| 60720 | 100 mg |
$595
|
10 days |
sulfo-Cyanine3.5 NHS ester
Amine-reactive activated ester of water-soluble, sulfonated sulfo-Cyanine3.5 dye.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 12320 | 1 mg |
$125
|
in stock | |
| 22320 | 5 mg |
$325
|
in stock | |
| 42320 | 25 mg |
$850
|
in stock | |
| 52320 | 50 mg |
$1490
|
in stock | |
| 62320 | 100 mg |
$1990
|
in stock |
sulfo-Cyanine5 NHS ester
Water-soluble sulfo-Cyanine5 succinimidyl ester for labeling various amine-containing molecules in the aqueous phase.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 13320 | 1 mg |
$91
|
in stock | |
| 23320 | 5 mg |
$119
|
in stock | |
| 43320 | 25 mg |
$345
|
in stock | |
| 53320 | 50 mg |
$557
|
in stock | |
| 63320 | 100 mg |
$890
|
in stock |
TAMRA NHS ester, 6-isomer
NHS-ester of TAMRA dye for proteins, peptides, and modified oligonucleotides labeling via reaction with amino groups. Pure 6-isomer.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 18120 | 1 mg |
$110
|
in stock | |
| 28120 | 5 mg |
$210
|
in stock | |
| 48120 | 25 mg |
$350
|
in stock | |
| 58120 | 50 mg |
$595
|
in stock | |
| 68120 | 100 mg |
$790
|
5 days |
TCO-PEG4-NHS ester (axial isomer)
A heterobifunctional linker with trans-cyclooctene and NHS ester groups flanking PEG4 (tetraethylene glycol) for labeling of antibodies, proteins, and other primary amine-containing biomolecules.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 3056-10mg | 10 mg |
$145
$137 |
in stock | |
| 3056-25mg | 25 mg |
$225
$215 |
in stock | |
| 3056-100mg | 100 mg |
$550
$522
|
in stock |
TR-X-NHS ester, 5-isomer
TR is a red-emitting fluorescent dye based on sulforhodamine scaffold, an amine-reactive NHS ester.
Show pricing
| Cat. # | Quantity | Price | Lead time | Buy this product |
|---|---|---|---|---|
| 1V220 | 1 mg |
$125
|
in stock | |
| 2V220 | 5 mg |
$325
|
in stock | |
| 4V220 | 25 mg |
$510
|
in stock | |
| 5V220 | 50 mg |
$895
|
in stock | |
| 6V220 | 100 mg |
$1490
|
in stock |
The count of items is incorrect.
Your cart is empty
$ 
