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Peptide, Oligonucleotide & Small Molecule Labeling Support
NHS Ester Labeling of Peptides, Oligonucleotides, and Small Molecules
NHS ester chemistry extends far beyond proteins and antibodies. Peptides, amino-modified oligonucleotides, amine-containing small molecules, and amino sugars all present primary amines that react with N-hydroxysuccinimide (NHS) esters under mild aqueous conditions to form stable amide bonds. This makes NHS ester dyes one of the most practical toolkits for building fluorescent probes, affinity tags, and assay reagents from molecular scaffolds that are very different from globular proteins.
This guide covers NHS ester labeling of peptides (N-terminus versus lysine side chain, solution-phase versus solid-phase conjugation, and degree of labeling control for receptor-binding peptides), amino-modified oligonucleotides (5-prime, 3-prime, and internal amino modifiers, bicarbonate buffer reactions, HPLC purification, and probe applications), small molecules (drugs, metabolites, and haptens carrying fluorescent tags or biotin), and related amine-containing biomolecules such as amino sugars. Site selection, purification, and characterization strategies for each molecular class are described so that researchers can plan conjugates that stay functional and well defined.
What Can BOC Sciences Help You Solve?
Labeling a peptide without losing receptor binding?
We help you choose between N-terminal and lysine labeling, control the degree of labeling, and verify activity after conjugation.
Need a fluorescent probe for qPCR, FISH, or microarrays?
We support amino-modified oligonucleotide design, dye coupling, and HPLC purification of labeled probes.
Building a tracer from a drug, metabolite, or hapten?
We help attach fluorescent tags or biotin to amine-containing small molecules and confirm the product by mass spectrometry.
Unsure which dye or amino modifier fits your probe design?
Compare FAM, Cy3, Cy5, TAMRA, HEX, and other NHS ester formats for your target molecule and detection platform.
Need custom labeled conjugates delivered ready to use?
Our services cover labeling, purification, characterization, and quality control for peptides, oligonucleotides, and small molecules.
Overview: NHS Ester Labeling Beyond Proteins
NHS ester labeling is often associated with proteins and antibodies, but the same amine-reactive chemistry is the standard route for labeling peptides, amino-modified oligonucleotides, and amine-containing small molecules. Each of these molecule classes presents primary amines that react with N-hydroxysuccinimide esters under mild aqueous conditions to form stable amide bonds, releasing N-hydroxysuccinimide as a benign leaving group. The result is a covalent conjugate that retains its chemical identity while gaining a fluorescent dye, a biotin tag, or another functional handle.
The reactive amines differ by class. In peptides they are the N-terminal alpha-amine and lysine epsilon-amino groups. In oligonucleotides a primary amine is introduced deliberately through an amino modifier during synthesis, giving full control over dye placement. In small molecules, labeling requires an existing primary amine, which is why amine-containing drugs, metabolites, and haptens are the usual candidates.
Core reaction conditions are shared: an NHS ester dye in anhydrous DMSO or DMF is added to the biomolecule in an amine-free buffer at pH 8.0 to 8.5 and incubated for 1 to 2 hours at room temperature, protected from light. A 5 to 20 fold molar excess of dye drives the desired degree of labeling. The differences between classes appear in labeling site design, purification strategy, and the analytical method used to confirm the conjugate.
Core principle:
An NHS ester reacts selectively with unprotonated primary amines to form a stable amide bond. The reaction competes with hydrolysis of the ester in
water, so pH, buffer choice, reagent handling, and timing must be controlled. For peptides, oligonucleotides, and small molecules, the same chemistry
applies, but the labeling site is usually more deliberate than in protein labeling, and the conjugate is smaller, which makes purification and
characterization correspondingly more precise. For a general introduction to the reagents and reaction conditions, see the
practical guide to NHS ester reagents for fluorescent labeling,
and browse the NHS ester reagents product range. These workflows sit within the broader
field of bioconjugation, which connects fluorescent probes to molecules across all size
scales.
Peptide Labeling: N-Terminus, Lysine Side Chains, and Labeling Position
Peptides present two natural amine handles: the N-terminal alpha-amine and lysine epsilon-amino groups. Choosing between them, and between solution and solid-phase labeling, determines dye position, degree of labeling, and how well the peptide keeps its biological function.
N-terminal labeling:
The N-terminal alpha-amine has a lower pKa than lysine side chains and is the primary reactive site in short or lysine-free peptides at pH 8.0 to 8.5. It produces a single, defined attachment point with minimal perturbation.
The N-terminal alpha-amine has a lower pKa than lysine side chains and is the primary reactive site in short or lysine-free peptides at pH 8.0 to 8.5. It produces a single, defined attachment point with minimal perturbation.
Lysine side chain labeling:
Each lysine epsilon-amino group can react with an NHS ester. In lysine-containing peptides, multiple dyes may attach, raising brightness but changing charge and isoelectric point, which can alter solubility and receptor interaction.
Each lysine epsilon-amino group can react with an NHS ester. In lysine-containing peptides, multiple dyes may attach, raising brightness but changing charge and isoelectric point, which can alter solubility and receptor interaction.
Solution-phase labeling:
Purified peptides are dissolved in bicarbonate or phosphate buffer and mixed with the NHS ester dye. This is the most flexible route and accepts any dye supplied as an NHS ester.
Purified peptides are dissolved in bicarbonate or phosphate buffer and mixed with the NHS ester dye. This is the most flexible route and accepts any dye supplied as an NHS ester.
Solid-phase labeling:
The dye can also be coupled on-resin using an orthogonally protected or selectively deprotected amine, giving a site-specific conjugate and simplifying removal of excess dye.
The dye can also be coupled on-resin using an orthogonally protected or selectively deprotected amine, giving a site-specific conjugate and simplifying removal of excess dye.
Degree of labeling for receptor-binding peptides:
For receptor-binding peptides, a degree of labeling (DOL) of 0.5 to 1.5 is usually recommended. A DOL near 1 means about one dye per peptide, balancing signal strength against the risk of blocking receptor-contact residues.
For receptor-binding peptides, a degree of labeling (DOL) of 0.5 to 1.5 is usually recommended. A DOL near 1 means about one dye per peptide, balancing signal strength against the risk of blocking receptor-contact residues.
Dye selection:
NHS ester versions of FITC, FAM, TAMRA, rhodamine, and cyanine dyes cover green, orange, and far-red windows. Consider sulfo-NHS variants for peptides with limited solubility.
NHS ester versions of FITC, FAM, TAMRA, rhodamine, and cyanine dyes cover green, orange, and far-red windows. Consider sulfo-NHS variants for peptides with limited solubility.
| Strategy | Reactive Amine | Effect on Activity | Best For |
|---|---|---|---|
| N-terminal labeling | N-terminal alpha-amine | Single defined site, usually well tolerated | Short peptides and receptor-binding peptides needing one label |
| Lysine epsilon-amino labeling | epsilon-amino group of lysine | Multiple sites possible; may block binding residues | Longer peptides and brighter multi-label conjugates |
| Acetylated N-terminus plus single lysine | One remaining lysine amine | Restricts labeling to one defined position | Strict 1:1 labeling with a controlled site |
| Solid-phase on-resin labeling | Orthogonally deprotected amine | Site-specific with easy dye removal | Complex sequences where solution purification is difficult |
Researchers who want an alternative amine-reactive route for peptide labeling may also consider isothiocyanate chemistry, which reacts with the same primary amines through a different linkage. Review fluorescein isothiocyanate dyes for amine labeling.
Oligonucleotide Labeling with Amino-Modified Bases
Unlike peptides, standard oligonucleotides do not carry convenient primary amines. A reactive amine is introduced during synthesis through an amino modifier, and the NHS ester dye is coupled to that handle after deprotection and purification, giving precise control over label position.
5-prime amino modifier C6:
A primary amine on a six-carbon spacer is added at the 5-prime terminus during synthesis. This is the most common format for fluorescent qPCR probes and FISH probes because it leaves the 3-prime end free for extension or quencher placement.
A primary amine on a six-carbon spacer is added at the 5-prime terminus during synthesis. This is the most common format for fluorescent qPCR probes and FISH probes because it leaves the 3-prime end free for extension or quencher placement.
3-prime amino modifier:
An amine at the 3-prime terminus supports immobilization on amine-reactive surfaces and microarray slides, and allows a quencher or second label at the opposite end.
An amine at the 3-prime terminus supports immobilization on amine-reactive surfaces and microarray slides, and allows a quencher or second label at the opposite end.
Internal amino-modified bases:
A modified nucleobase carrying a primary amine can be placed internally, which is useful for dual-labeled probes where dye and quencher bracket a central region.
A modified nucleobase carrying a primary amine can be placed internally, which is useful for dual-labeled probes where dye and quencher bracket a central region.
Reaction in bicarbonate buffer:
Amino-modified oligonucleotides are dissolved in 0.1 M sodium bicarbonate at pH 8.5 and reacted with a 10 to 20 fold molar excess of NHS ester dye for 1 to 2 hours. Avoid Tris and other primary amine buffers.
Amino-modified oligonucleotides are dissolved in 0.1 M sodium bicarbonate at pH 8.5 and reacted with a 10 to 20 fold molar excess of NHS ester dye for 1 to 2 hours. Avoid Tris and other primary amine buffers.
Purification by HPLC:
Reverse-phase HPLC separates the labeled probe from unlabeled oligonucleotide and free dye, which is essential because quantitative assays need a single fluorescent species.
Reverse-phase HPLC separates the labeled probe from unlabeled oligonucleotide and free dye, which is essential because quantitative assays need a single fluorescent species.
Probe applications:
Amino-modified probes labeled with FAM, Cy3, Cy5, HEX, or TAMRA support qPCR detection, FISH imaging, and microarray capture. Far-red cyanine dyes are common on array platforms for brightness and spectral separation.
Amino-modified probes labeled with FAM, Cy3, Cy5, HEX, or TAMRA support qPCR detection, FISH imaging, and microarray capture. Far-red cyanine dyes are common on array platforms for brightness and spectral separation.
| Modifier Position | Handle | Typical Applications | Design Note |
|---|---|---|---|
| 5-prime amino modifier C6 | Primary amine on a C6 spacer | Fluorescent qPCR probes, FISH probes | Keeps the 3-prime end available for extension or quencher. |
| 3-prime amino modifier | Primary amine on a carbon spacer | Surface immobilization, microarray capture probes | Useful when the probe is fixed to a solid support. |
| Internal amino-modified base | Amine on a modified nucleobase | Dual-labeled FRET probes, internal dual-label constructs | Place the label away from the hybridization region if possible. |
| Amino modifier with longer spacer | Extended carbon linker | Crowded dual-label probes | Reduces dye and quencher interaction when labels are close. |
For assay design around labeled probes, the same dye families support detection across qPCR, FISH, microarray analysis, and molecular diagnostics. When choosing fluorophores for multiplex nucleic acid assays, cyanine dyes for fluorescent labeling provide matched excitation and emission profiles.
Small Molecule Labeling: Drugs, Metabolites, Haptens, and Amino Sugars
Small molecules can be conjugated to fluorescent dyes or biotin only when they carry a reactive primary amine. Amine-containing drugs, drug metabolites, haptens, and amino sugars are therefore the natural candidates for NHS ester labeling, and the resulting conjugates serve as tracers, immunogens, and assay reagents.
Amine-containing drugs and metabolites:
Compounds with a primary amine, such as many aminoglycosides, amino acid derivatives, and active metabolites, react directly with NHS ester dyes. The labeled product can be used as a fluorescent tracer in binding and uptake studies.
Compounds with a primary amine, such as many aminoglycosides, amino acid derivatives, and active metabolites, react directly with NHS ester dyes. The labeled product can be used as a fluorescent tracer in binding and uptake studies.
Hapten conjugation:
Small molecules are often conjugated to carrier proteins through NHS ester chemistry, but a fluorescent or biotin label can also be attached directly to the hapten when it carries an amine, producing a defined small molecule conjugate.
Small molecules are often conjugated to carrier proteins through NHS ester chemistry, but a fluorescent or biotin label can also be attached directly to the hapten when it carries an amine, producing a defined small molecule conjugate.
Fluorescent tags for assay development:
Attaching dyes such as FITC, FAM, TAMRA, Cy3, or Cy5 to a small molecule creates a tracer that can be displaced in competition assays or tracked by fluorescence polarization, flow cytometry, or imaging.
Attaching dyes such as FITC, FAM, TAMRA, Cy3, or Cy5 to a small molecule creates a tracer that can be displaced in competition assays or tracked by fluorescence polarization, flow cytometry, or imaging.
Biotin conjugation:
Biotin-NHS esters attach a biotin tag to amine-containing small molecules, enabling capture on streptavidin supports for pull-down, enrichment, and detection workflows.
Biotin-NHS esters attach a biotin tag to amine-containing small molecules, enabling capture on streptavidin supports for pull-down, enrichment, and detection workflows.
Amino sugars and carbohydrates:
Amino sugars such as glucosamine and its derivatives carry a primary amine and can be labeled with NHS ester dyes for glycan tracking and lectin binding studies. Other aminated carbohydrates can be conjugated in the same way.
Amino sugars such as glucosamine and its derivatives carry a primary amine and can be labeled with NHS ester dyes for glycan tracking and lectin binding studies. Other aminated carbohydrates can be conjugated in the same way.
Characterization by mass spectrometry:
Because small molecule conjugates have defined masses, ESI-MS or high-resolution MS confirms that the mono-labeled product formed without di-addition or hydrolysis side products.
Because small molecule conjugates have defined masses, ESI-MS or high-resolution MS confirms that the mono-labeled product formed without di-addition or hydrolysis side products.
Practical note:
Small molecule labeling is small scale, typically micromolar, and the dye excess is often higher than for peptides to push the reaction to
completion. Keep the volume small, use fresh NHS ester, and purify promptly to limit hydrolysis and photo-bleaching.
Site Selection: Protecting Critical Residues
The biological activity of a labeled peptide or small molecule depends on where the dye is attached. Because NHS esters modify every accessible primary amine, the labeling site must be chosen with knowledge of residues required for binding or function.
Map the functional sequence first:
Review the structure-activity relationship to identify residues that contact the receptor. Lysines inside the binding motif should be avoided as labeling sites.
Review the structure-activity relationship to identify residues that contact the receptor. Lysines inside the binding motif should be avoided as labeling sites.
Prefer the N-terminus for single labels:
For many short receptor-binding peptides, the N-terminus is not part of the pharmacophore, so an N-terminal label preserves binding better than a label at an internal lysine.
For many short receptor-binding peptides, the N-terminus is not part of the pharmacophore, so an N-terminal label preserves binding better than a label at an internal lysine.
Use a spacer to reduce steric interference:
Amino modifiers with six-carbon or longer spacers move the dye away from the peptide backbone, reducing steric clash with the receptor and improving recognition.
Amino modifiers with six-carbon or longer spacers move the dye away from the peptide backbone, reducing steric clash with the receptor and improving recognition.
Block unwanted amines:
If multiple lysines exist but a single defined label is needed, the N-terminus can be acetylated and lysine side chains protected or removed so that only one amine remains reactive.
If multiple lysines exist but a single defined label is needed, the N-terminus can be acetylated and lysine side chains protected or removed so that only one amine remains reactive.
Control the molar ratio:
Lower dye-to-peptide ratios bias the reaction toward mono-labeling, while higher ratios produce multiple labels. For receptor-binding studies, keep the degree of labeling between 0.5 and 1.5.
Lower dye-to-peptide ratios bias the reaction toward mono-labeling, while higher ratios produce multiple labels. For receptor-binding studies, keep the degree of labeling between 0.5 and 1.5.
Verify activity after labeling:
Measure binding or functional activity of the conjugate against the unlabeled peptide. If activity drops, move the label or reduce the degree of labeling.
Measure binding or functional activity of the conjugate against the unlabeled peptide. If activity drops, move the label or reduce the degree of labeling.
Purification and Characterization by Molecule Class
Purification removes unreacted dye and unlabeled starting material, while characterization confirms that the correct conjugate formed with an appropriate degree of labeling. Methods differ by class because conjugates differ in size, charge, and spectral properties.
| Molecule Class | Purification | Characterization | Key Checks |
|---|---|---|---|
| Peptide conjugates | Reverse-phase HPLC, desalting | ESI-MS or MALDI-TOF MS, UV absorbance | Correct mass, degree of labeling, no residual free dye |
| Oligonucleotide conjugates | Reverse-phase or ion-exchange HPLC | Denaturing PAGE, ESI-MS, UV absorbance ratio | Single labeled species, dye integrity, correct probe length |
| Small molecule conjugates | HPLC, flash chromatography | ESI-MS or HRMS, NMR, HPLC purity | Correct labeled mass, no di-addition or hydrolysis products |
| Amino sugar conjugates | HPLC, size-exclusion | MS, colorimetric sugar assays | Labeling at the amino group, hydrolytic stability |
Degree of labeling by absorbance:
For peptide and oligonucleotide conjugates, DOL is estimated from absorbance at 280 nm and at the dye maximum, correcting for dye contribution at 280 nm.
For peptide and oligonucleotide conjugates, DOL is estimated from absorbance at 280 nm and at the dye maximum, correcting for dye contribution at 280 nm.
Mass spectrometry:
ESI-MS confirms the mass of small molecule and peptide conjugates and distinguishes mono-labeled from multi-labeled species. HRMS adds confidence for novel small molecule conjugates.
ESI-MS confirms the mass of small molecule and peptide conjugates and distinguishes mono-labeled from multi-labeled species. HRMS adds confidence for novel small molecule conjugates.
Denaturing PAGE for oligonucleotides:
Denaturing polyacrylamide gel electrophoresis resolves labeled probes from unlabeled and doubly labeled species, and it is the standard visual check for probe quality. Related gel electrophoresis resources cover running conditions for nucleic acid analysis.
Denaturing polyacrylamide gel electrophoresis resolves labeled probes from unlabeled and doubly labeled species, and it is the standard visual check for probe quality. Related gel electrophoresis resources cover running conditions for nucleic acid analysis.
Free dye removal:
Unreacted dye must be removed completely because it raises background. Desalting, size-exclusion, HPLC, and ethanol precipitation are used depending on the class and dye.
Unreacted dye must be removed completely because it raises background. Desalting, size-exclusion, HPLC, and ethanol precipitation are used depending on the class and dye.
Need Help Choosing a Labeling Route for Your Peptide, Oligonucleotide, or Small Molecule?
BOC Sciences can support labeling position design, dye selection, NHS ester conjugation, purification, and characterization for peptides, amino-modified oligonucleotides, amine-containing small molecules, and amino sugars.
Request Peptide and Small Molecule Labeling SupportA Practical Workflow for Choosing a Labeling Route
The right labeling route depends on the molecule class, the available amine handles, and the intended application. The steps below move from defining the target to delivering a purified, characterized conjugate.
Step 1: Identify the reactive amine handle
Determine whether the molecule carries a primary amine. Peptides use the N-terminus or lysine side chains, oligonucleotides need an amino modifier, and small molecules must already contain an amine.
Determine whether the molecule carries a primary amine. Peptides use the N-terminus or lysine side chains, oligonucleotides need an amino modifier, and small molecules must already contain an amine.
Step 2: Choose the labeling position
Select a site that does not interfere with receptor binding or function. For peptides, compare N-terminal and lysine labeling. For oligonucleotides, pick 5-prime, 3-prime, or internal modification based on the probe architecture.
Select a site that does not interfere with receptor binding or function. For peptides, compare N-terminal and lysine labeling. For oligonucleotides, pick 5-prime, 3-prime, or internal modification based on the probe architecture.
Step 3: Select the dye and NHS ester format
Match the dye to the platform: FAM or FITC for green channels, Cy3 or TAMRA for orange, Cy5 for far-red, and HEX for qPCR reporters. Consider sulfo-NHS variants for aqueous solubility.
Match the dye to the platform: FAM or FITC for green channels, Cy3 or TAMRA for orange, Cy5 for far-red, and HEX for qPCR reporters. Consider sulfo-NHS variants for aqueous solubility.
Step 4: Run the conjugation reaction
Dissolve the NHS ester in anhydrous DMSO, add a 5 to 20 fold molar excess to the molecule in bicarbonate or phosphate buffer at pH 8.0 to 8.5, and incubate for 1 to 2 hours at room temperature protected from light.
Dissolve the NHS ester in anhydrous DMSO, add a 5 to 20 fold molar excess to the molecule in bicarbonate or phosphate buffer at pH 8.0 to 8.5, and incubate for 1 to 2 hours at room temperature protected from light.
Step 5: Purify the conjugate
Use HPLC for oligonucleotides and most peptide and small molecule conjugates, with desalting or size-exclusion to remove residual dye. Confirm separation by absorbance at the dye maximum.
Use HPLC for oligonucleotides and most peptide and small molecule conjugates, with desalting or size-exclusion to remove residual dye. Confirm separation by absorbance at the dye maximum.
Step 6: Characterize and validate
Confirm the mass by MS, estimate the degree of labeling by UV absorbance, and verify that the conjugate retains its functional activity before it is used in assays.
Confirm the mass by MS, estimate the degree of labeling by UV absorbance, and verify that the conjugate retains its functional activity before it is used in assays.
Common Challenges in Non-Protein NHS Ester Labeling
Each conjugate class has characteristic failure modes. Recognizing them early improves yield, reduces background, and keeps the conjugate functional.
Over-labeling reduces activity
Excess dye or multiple lysine sites can block receptor-binding residues. Keep DOL between 0.5 and 1.5 for functional peptides and confirm activity after conjugation.
Hydrolysis at high pH
Working above pH 8.5 accelerates NHS ester hydrolysis, lowering yield. Keep reactions near pH 8.0 to 8.5 and use freshly dissolved reagent to reduce water competition.
Amine buffers compete for the dye
Tris, glycine, and ammonium salts react with NHS esters and reduce labeling efficiency. Exchange the molecule into bicarbonate, phosphate, borate, or HEPES buffer before conjugation.
Incomplete removal of free dye
Residual dye inflates background in qPCR, FISH, and imaging. Use HPLC or repeated desalting and verify removal by absorbance or fluorescence scanning.
Low yield for poorly soluble small molecules
Hydrophobic drugs and metabolites may precipitate when dye dissolved in DMSO is added. Use a sulfo-NHS dye, raise the aqueous solvent fraction, or pre-dissolve the compound in a compatible co-solvent.
Mixtures of labeled oligonucleotide species
Incomplete amino modification during synthesis produces a mix of unlabeled, singly, and doubly labeled probes. HPLC purification and denaturing PAGE QC deliver a single defined species for quantitative assays.
BOC Sciences Labeling Services for Peptides, Oligonucleotides, and Small Molecules
BOC Sciences supports the full range of non-protein NHS ester labeling, from reagent supply and labeling position design to conjugate synthesis, purification, and quality control. Custom programs can also be connected with fluorescent labeling services for larger or multi-partner projects.
Peptide Fluorescent Labeling
Custom labeling of research peptides at the N-terminus or lysine side chains with defined dye placement.
- N-terminal and lysine labeling
- Solution-phase and solid-phase conjugation
- Degree of labeling control from 0.5 to 1.5
- HPLC purification of labeled peptides
Oligonucleotide Probe Labeling
Coupling of NHS ester dyes to amino-modified oligonucleotides for quantitative and imaging assays.
- 5-prime, 3-prime, and internal amino modification
- FAM, Cy3, Cy5, TAMRA, HEX, and other dye coupling
- Reverse-phase HPLC purification
- qPCR, FISH, and microarray probe delivery
Small Molecule Tracer and Hapten Synthesis
Attachment of fluorescent tags or biotin to amine-containing drugs, metabolites, and haptens.
- Fluorescent tracer preparation
- Biotin conjugation for capture assays
- Hapten and immunogen conjugate synthesis
- MS and HPLC characterization
Labeling Position Design
Structure-guided selection of the labeling site to preserve receptor binding and biological activity.
- Structure-activity relationship review
- Protecting critical residues
- N-terminal versus lysine placement
- Activity verification after labeling
Amino Sugar and Carbohydrate Labeling
NHS ester conjugation of amino sugars and aminated carbohydrates for glycan tracking and binding studies.
- Amino sugar fluorescent labeling
- Biotin tagging of aminated glycans
- HPLC purification
- Mass-based conjugate confirmation
Custom Dye Reagent and Modifier Supply
Access to NHS ester dyes, amino modifiers, and custom dye synthesis to support in-house labeling programs.
- NHS ester dye supply
- Amino modifier support
- Custom dye and linker synthesis
- Scaled reagent production
Start Your Non-Protein Labeling Project with BOC Sciences
Whether you need a labeled receptor-binding peptide, a fluorescent oligonucleotide probe for qPCR or FISH, a small molecule tracer, or a custom amino sugar conjugate, BOC Sciences can help you select the labeling site, dye, and reaction conditions and deliver a purified, characterized product.
Send Your Labeling RequirementsRecommended NHS Ester Dyes and Amine-Reactive Reagents for Non-Protein Labeling
The following products are commonly used in peptide, oligonucleotide, and small molecule labeling projects. The list includes NHS ester derivatives and fluorescent dyes supplied in amine-reactive formats that are suitable for building fluorescent probes and tracers.
| Catalog | Product Name | CAS | Inquiry |
|---|---|---|---|
| F01-0166 | BODIPY 493/503 NHS Ester | 216961-98-7 | Bulk Inquiry |
| A16-0170 | Rhodamine-123 | 62669-70-9 | Bulk Inquiry |
| A16-0033 | 6-Carboxyfluorescein | 3301-79-9 | Bulk Inquiry |
| A16-0036 | Calcein Blue | 54375-47-2 | Bulk Inquiry |
| A16-0093 | Rhodamine 6G | 989-38-8 | Bulk Inquiry |
| A16-0003 | Phalloidin-TFAX 488 | 289620-19-5 | Bulk Inquiry |
| A16-0002 | Phalloidin-TRITC | 915013-10-4 | Bulk Inquiry |
| A01-0005 | Rhodamine B | 81-88-9 | Bulk Inquiry |
| R12-0001 | BODIPY 493/503 | 121207-31-6 | Bulk Inquiry |
| F06-0011 | Coumarin 153 | 53518-18-6 | Bulk Inquiry |
| F03-0001 | Sulfo-Cyanine3 amine | 2183440-43-7 | Bulk Inquiry |
| A19-0102 | SYBR Green II | 195199-08-7 | Bulk Inquiry |
Explore More NHS Ester Resources
Explore related guides on NHS ester reactivity, fluorophore selection, substrate-specific labeling, and conjugation chemistry. These resources can help you evaluate reactive groups, reaction conditions, reagent properties, and alternative strategies for labeling diverse molecular substrates.
- NHS Ester Reaction Mechanism: Acylation of Primary Amines and Hydrolysis Kinetics
- How to Choose the Right NHS Ester Dye for Your Labeling Experiment
- NHS Ester vs Maleimide vs Click Chemistry: Choosing the Right Conjugation Chemistry
- What Are NHS Ester Reagents? Chemistry, Reactivity, and Role in Fluorescent Labeling
- NHS Ester Crosslinkers for Bioconjugation: Homo- and Heterobifunctional Reagents
- Custom NHS Ester Synthesis and Fluorescent Conjugation Services
Frequently Asked Questions
These questions address common decision points in NHS ester labeling of peptides, oligonucleotides, and small molecules, from position selection to purification and quality control.
How do I label a peptide without losing its biological activity?
Choose a labeling position outside the receptor-binding region. For most short peptides the N-terminus is well tolerated, while lysines inside the active motif should be avoided. Keep the degree of labeling between 0.5 and 1.5, use a spacer, and measure binding after conjugation to confirm function was retained.
Why are oligonucleotides amino-modified before NHS ester labeling?
Standard DNA and RNA strands have no primary amines for NHS ester coupling. An amino modifier introduced during solid-phase synthesis, such as a 5-prime amino modifier C6 or an internal amino-modified base, provides the reactive handle for defined dye placement.
What is the difference between 5-prime and 3-prime amino modification?
A 5-prime amino modifier places the amine at the start of the sequence, ideal for fluorescent reporters in qPCR probes because the 3-prime end stays free for extension or a quencher. A 3-prime modifier places the amine at the end and is used for surface immobilization or attaching a quencher at the opposite end.
Which buffer should I use for labeling amino-modified oligonucleotides?
Use an amine-free buffer such as 0.1 M sodium bicarbonate at pH 8.5. Tris, glycine, and ammonium salts contain primary amines that compete for the NHS ester and should be removed by ethanol precipitation or buffer exchange first.
How is the degree of labeling measured for peptides and small molecules?
The degree of labeling is estimated from UV absorbance using the extinction coefficients of the biomolecule and dye, or confirmed by mass spectrometry, which distinguishes unlabeled, mono-labeled, and multi-labeled species. HRMS and NMR provide the highest confidence for small molecules.
Request Labeling Support for Your Peptide, Oligonucleotide, or Small Molecule
Share your target molecule, dye preferences, and assay goals with BOC Sciences. Our team can help you design the labeling site, select suitable NHS ester reagents, and deliver a purified, characterized conjugate.
Labeling position design
Compare N-terminal, lysine, 5-prime, 3-prime, and internal labeling strategies for your molecule.
Compare N-terminal, lysine, 5-prime, 3-prime, and internal labeling strategies for your molecule.
Dye and reagent selection
Match FAM, Cy3, Cy5, TAMRA, HEX, and other NHS ester dyes to your detection platform.
Match FAM, Cy3, Cy5, TAMRA, HEX, and other NHS ester dyes to your detection platform.
Conjugate purification and QC
HPLC, MS, and denaturing PAGE support for defined single-species conjugates.
HPLC, MS, and denaturing PAGE support for defined single-species conjugates.
Bulk product inquiry
Request pricing, availability, packaging, and project-specific supply information.
Request pricing, availability, packaging, and project-specific supply information.
