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DyLight™ 680 NHS-Ester (Thermo Scientific™)

Thermo Scientific DyLight 680 Amine-Reactive Dye is an NHS ester-activated derivative of high-performance DyLight 680 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods.

DyLight 680 produces near-infrared (IR) fluorescence that replaces other near-IR dyes, including Cy5.5™ dye and Alexa Fluor™ 680, and is ideal for multi-color applications. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 680 Amine-Reactive Dye is also available as part of two antibody labeling kit sizes.

Features of DyLight 680 NHS-Ester:

High performance—DyLight 680 fluoresces brighter than Alexa Fluor 680 and Cy5.5 dye
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting, or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 680 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 680 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

We also offer Standard and Microscale DyLight 680 Antibody Labeling Kits for fast and efficient fluorescent labeling of antibodies for use in fluorescence methods.The standard size kit contains all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG. Both kit sizes include the Amine-Reactive DyLight 680 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

Related Products
DyLight™ 680 Antibody Labeling Kit
DyLight™ 680 Microscale Antibody Labeling Kit

DyLight™ 755 Microscale Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 755 Microscale Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 755 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG—the amine-reactive DyLight 755 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 755 is a near-IR fluor that is invisible to the naked eye but increases the staining options when using infrared imaging systems. DyLight 755 has spectral properties that are very similar to other near-IR dyes, including Alexa Fluor™ 750. The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 755 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 755 NHS Ester:

High performance—DyLight 755 replaces Alexa Fluor 755 for near-infrared staining
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Convenient kit sizes—standard and microscale sizes are offered to match your experimental needs
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

Applications:
• Primary antibody labeling for immunofluorescence microscopy, immunohistochemistry (IHC), Western blotting, or ELISA assay
• Target protein labeling for in vitro and in vivo fluorescent detection strategies

DyLight 755 Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 755 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

Related Products
DyLight™ 755 NHS Ester
DyLight™ 755 Antibody Labeling Kit

Biotin-4-Fluorescein (Invitrogen™)

Biotin-4-fluorescein can be used to quantitate available biotin-binding sites or the concentration of avidin or streptavidin through the strong quenching associated when this molecule binds to avidin.

DyLight™ 650-4xPEG NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 650-4xPEG Amine-Reactive Dye is a derivative of our high-performance DyLight 650 Dye that can be used to fluorescently label antibodies and other proteins for far-red detection.

The DyLight 650-4xPEG dye contains 4 polyethylene glycol (PEG) chains that are non-cytotoxic, enhance fluorescence, and reduce nonspecific binding of conjugates made with them. Conjugates made with DyLight 650-4xPEG Dye can be used as molecular probes for cellular imaging and other fluorescence detection methods. The far-red to NIR fluorescence properties of DyLight 650-4xPEG Dye make it especially useful in a variety of biological, chemical, and pharmaceutical applications, including in vivo imaging. The PEG chains also improve solubility of the dyes and labeled molecules in aqueous solution, aid in cell permeability, and improve tissue retention.

Features of DyLight 650-4xPEG NHS Ester:

High fluorescence intensity—significantly brighter fluorescence than Alexa Fluor™ 647
PEGylated—improves solubility in aqueous solution and aids in cell permeability

Applications:
• Fluorescence microscopy
In vivo or ex vivo imaging
• Cell-based assays
• Flow cytometry/fluorescence-activated cell sorting (FACS)

DyLight 650-4xPEG Amine-Reactive Dye is activated with an N-hydroxysuccinimide (NHS) ester moiety to react with exposed N-terminal α-amino groups or the ε-amino groups of lysine residues to form stable amide bonds. Learn more about NHS ester chemistry.

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to the labeling reagent.

R-Phycoerythrin (Invitrogen™)

Phycobiliprotein bioconjugates including antibody and other protein conjugates can be made with the exceptionally bright phycobiliprotein, R-phycoerythrin using the Protein-Protein Crosslinking Kit (P6305).

Alexa Fluor™ 546 NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 546 is a bright yellow dye. Used in imaging and flow cytometry, Alexa Fluor® 546 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 546 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

The NHS ester (or succinimidyl ester) of Alexa Fluor® 546 is the most popular tool for conjugating this dye to a protein or antibody. NHS esters can be used to label to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting Alexa Fluor® conjugate will exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® NHS ester:

Fluorophore label: Alexa Fluor® 546 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 554/570 nm
Extinction coefficient: 112,000 cm-1M-1
Molecular weight: 1159.6

Typical Conjugation Reaction
You can conjugate amine-reactive reagents with virtually any protein or peptide (the provided protocol is optimized for IgG antibodies). You can scale the reaction for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The Alexa Fluor® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO) (D12345), and the reaction is carried out in 0.1–0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free Alexa Fluor® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

BODIPY™ 558/568 NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® 558/568 dye is bright, red fluorescent dye. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® 558/568 dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® 558/568 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

The NHS ester (or succinimidyl ester) of BODIPY® 558/568 is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® 558/568 conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

Detailed information about this BODIPY® 558/568 NHS ester:

Fluorophore label: BODIPY® 558/568 dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 559/568 nm
Extinction coefficient: 97,000 cm-1M-1
Molecular weight: 443.23

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and the reaction is carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free BODIPY® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

GlycanAssure™ AutoXpress Kit with CE Module

The GlycanAssure AutoXpress Kit is designed for use on the AutoMate Express System for GlycanAssure AutoXpress Kits, for hands-free automation of the preparation of labelled glycans from glycoprotein samples. The kit includes GlycanAssure AutoXpress cartridges that contain pre-filled reagents and plastic tubes and tips to perform hands-free glycan release and labelling of up to 13 samples in one run. This version of the kit contains a CE module with additional chemistries needed for CE analysis.

The GlycanAssure AutoXpress cartridge reagents are based on the established GlycanAssure chemistry for N-glycan rapid release, labeling, and cleanup that applies a novel method for N-glycan release in a complete form. The GlycanAssure APTS N-glycan sample prep method consists of rapid deglycosylation using PNGase-F enzyme followed by magnetic bead-based glycan purification, glycan labeling with ATPS dye, and excess dye removal. The GlycanAssure AutoXpress Kit combined with the AutoMate Express System offers a single, automated N-glycan sample prep workflow for both high throughput (CE) and characterization (UHPLC) biopharma applications.

Key features of the combined kit/system include:
• Fully automated sample prep for N-glycan analysis
• Rapid results with cartridge-based reagents for glycan release and labelling
• High quality glycan data with reduced analyst error
• Labeled glycans can be analyzed on LC or CE platforms
• Small system footprint and low-cost automation platform make it adaptable in both development and QC labs
• Consistent data for easy method development and transfer across the globe

Pierce™ Streptavidin, Hydrazide-Activated (Thermo Scientific™)

Thermo Scientific Pierce Hydrazide-Activated Streptavidin conjugate include recombinant streptavidin in a purified form activated for crosslinking to carbonyl groups in a molecule.

Related Products
Pierce™ Streptavidin
Pierce™ Streptavidin, Horseradish Peroxidase Conjugated
Pierce™ Streptavidin, Alkaline Phosphatase Conjugated
Pierce™ Streptavidin, Maleimide-Activated

Alexa Fluor™ 488 Hydrazide (Invitrogen™)

Alexa Fluor® 488 Hydrazide is useful as a cell tracer and as a reactive dye for labeling aldehydes or ketones in polysaccharides or glycoproteins. Alexa Fluor® 488 is a bright, green fluorescent dye with excitation ideally suited to the 488 nn laser line. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 488 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 488 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection (learn more).

Detailed information about this AlexaFluor® hydrazide:

• Fluorophore label : Alexa Fluor® 488 dye
• Reactive group: hydrazide
• Reactivity: Aldehydes or keytones in polysaccharides or glycoproteins
• Ex/Em of the conjugate: 493/517 nm
• Extinction coefficient: 71,000 cm-1M-1
• Spectrally similar dyes: FITC, GFP
• Molecular weight: 570.48

Cell Tracking and Tracing Applications
Alexa Fluor® hydrazides and hydroxlamines are useful as low molecular weight, membrane-impermeant, aldehyde-fixable cell tracers, exhibiting brighter fluorescence and greater photostability than cell tracers derived from other spectrally similar fluorophores. They are easily loaded into cells by microinjection, infusion from patch pipette, or uptake induced by our Influx™ Pinocytic Cell-Loading Reagent. Learn more about cell tracking and tracing.

Glycoprotein and Polysaccharide Labeling Applications
The Alexa Fluor® hydrazides and hydroxlamines are reactive molecules that can be used to add a fluorescent label to biomolecules containing aldehydes or ketones. Aldehydes and ketones can be introduced into polysaccharides and glycoproteins by periodate-mediated oxidation of vicinal diols. Galactose oxidase can also be used to oxidize terminal galactose residues of glycoproteins to aldehydes.

Hydrazide vs Hydroxylamine
Hydrazine derivatives react with ketones and aldehydes to yield relatively stable hydrazones. Hydroxylamine derivatives (aminooxy compounds) react with aldehydes and ketones to yield oximes. Oximes are superior to hydrazones with respect to hydrolytic stability. Both hydrazones and oximes can be reduced with sodium borohydride (NaBH4) to further increase the stability of the linkage.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

Related Products
DMSO (dimethylsulfoxide) (D12345)
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

CellTracker™ Blue CMHC Dye (Invitrogen™)

CellTracker™ Blue CMHC (4-chloromethyl-7-hydroxycoumarin) is a fluorescent dye well suited for monitoring cell movement or location. This dye is well retained, allowing for multigenerational tracking of cellular movements. And the blue excitation/emission spectra are ideal for multiplexing with green and red fluorescent dyes and proteins.

Need a different emission spectrum or longer tracking? View our other mammalian cell tracking products.

• Easy to use—remove medium, add dye, incubate 30 minutes, and image cells
• Fluorescent signal retention of >72 hours (typically three to six generations)
• Blue excitation/emission spectra (372/470 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

CellTracker™ Blue CMHC fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into cell membrane-impermeant reaction products. CellTracker™ Blue CMHC dye is retained in living cells through several generations. The dye is transferred to daughter cells, but not adjacent cells in a population. CellTracker™ Blue CMHC dye is designed to display fluorescence for at least 72 hours, and the dye exhibits ideal tracking properties: it is stable, nontoxic at working concentrations, well retained in cells, and brightly fluorescent at physiological pH. Additionally, the excitation and emission spectra of CellTracker™ Blue CMHC dye are well separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra allowing for multiplexing.

Dabcyl, SE, 4-((4-(dimethylamino)phenyl)azo)benzoic Acid, Succinimidyl Ester (Invitrogen™)

The amine-reactive quencher, dabcyl succinimidyl ester has a broad and intense visible absorption but no fluorescence making it useful as an acceptor in fluorescence resonance energy transfer (FRET) applications.

N-(1-Pyrene)Maleimide (Invitrogen™)

The thiol-reactive N-(1-pyrene)maleimide can be used to create environment-sensitive bioconjugates with this unique fluorophore.

GlcNAz (N-azidoacetylglucosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce GlcNAz (N-azidoacetylglucosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
ManNAz (N-azidoacetylmannosamine tetraacylated)
GalNAz (N-azidoacetylgalactosamine tetraacylated)

BODIPY™ R6G NHS Ester (Succinimidyl Ester) (Invitrogen™)

BODIPY® R6G dye is bright, yellow fluorescent dye with similar excitation and emission to rhodamine 6G. It has a high extinction coefficient and fluorescence quantum yield and is relatively insensitive to solvent polarity and pH change. In contrast to the highly water soluble fluorophores Alexa Fluor® 488 dye and fluorescein (FITC), BODIPY® dyes have unique hydrophobic properties ideal for staining lipids, membranes, and other lipophilic compounds. BODIPY® R6G dye has a relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization-based assays and a large two-photon cross-section for multiphoton excitation. In addition to reactive dye formulations, we offer BODIPY® R6G dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection.

The NHS ester (or succinimidyl ester) of BODIPY® R6G is the most popular tool for conjugating the dye to a protein or antibody. NHS esters can be used to label the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. The resulting BODIPY® R6G conjugates exhibit bright fluorescence, narrow emission bandwidths, and relatively long excited-state lifetimes, which can be useful for fluorescence polarization assays and two-photon excitation (TPE) microscopy.

This reactive dye contains a C3 alkyl spacer between the fluorophore and the NHS ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated.

Detailed information about this BODIPY® R6G NHS ester:

Fluorophore label: BODIPY® R6G dye
Reactive group: NHS ester (succinimidyl ester)
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 528/547 nm
Extinction coefficient: 70,000 cm-1M-1
Molecular weight: 437.21

Typical Conjugation Reaction
Amine-reactive reagents can be conjugated with virtually any protein or peptide; the provided protocol is optimized for IgG antibodies. The reaction can be scaled for any amount of protein, but the concentration of the protein should be at least 2 mg/mL for optimal results. We recommend trying three different degrees of labeling, using three different molar ratios of the reactive reagent to protein.

The BODIPY® NHS ester is typically dissolved in high-quality anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and the reaction is carried out in 0.1-0.2 M sodium bicarbonate buffer, pH 8.3, at room temperature for 1 hour. Because the pKa of the terminal amine is lower than that of the lysine epsilon-amino group, you may achieve more selective labeling of the amine terminus using a buffer closer to neutral pH.

Conjugate Purification
Labeled antibodies are typically separated from free BODIPY® dye using a gel filtration column, such as Sephadex™ G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration medium with an appropriate molecular weight cut-off or purify by dialysis. We offer several purification kits optimized for different quantities of antibody conjugate:
Antibody Conjugate Purification Kit for 0.5-1 mg (A33086)
Antibody Conjugate Purification Kit for 20-50 µg (A33087)
Antibody Conjugate Purification kit for 50-100 µg (A33088)

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits to fit your starting material and your experimental setup. See our Antibody Labeling kits or use our Labeling Chemistry Selection Tool for other choices. To learn more about our labeling kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in The Molecular Probes® Handbook.

We’ll Make a Custom Conjugate for You
If you can’t find what you’re looking for in our online catalog, we’ll prepare a custom antibody or protein conjugate for you. Our custom conjugation service is efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.