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Click-iT™ Amine sDIBO Alkyne Invitrogen™

Click-iT Amine sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce amine bioconjugates. sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less “sticky” and give lower signal background in biological samples. Copper-free Click bio-conjugation reactions are ideal for surface labeling of live cells and also minimize damage to enzymes and fluorescent proteins like GFP or R-PE. Macromolecules that have been azide-modified enzymatically, chemically, or metabolically can be now be labeled easily, yielding more soluble bioconjugates with improved biological labeling utility.

• More soluble than DIBO cyclooctynes leading to more soluble conjugates
• Minimal background potential in cells and tissues compared to original DIBO cycloctynes

Pierce™ Biotin Thermo Scientific™

Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells and is critical for a number of biological processes. The valeric acid side chain of the biotin molecule can be derivatized in order to incorporate various reactive groups that are used to attach biotin to other molecules. In the context of immunohistochemistry (IHC), biotin is conjugated to antibodies or to the enzyme reporters used to detect target antigens.

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Pierce™ Biotin-Fluorescein Conjugate

Alexa Fluor™ 488 5-SDP Ester (Alexa Fluor™ 488 Sulfodichlorophenol Ester) Invitrogen™

The SDP (sulfodicholorphenol) ester is currently the most hydrolytically stable amine-reactive moiety available from Invitrogen. Conjugates produced with the Alexa Fluor® 488 5-SDP ester produce the same strong amide bond between the dye and the compound of interest as succinimidyl (SE or NHS) and tetrafluorophenyl (TFP) esters, but with the improved stability in water and buffers, the SDP ester can potentially offer increased control and consistency in these reactions compared to the NHS and TFP esters.

TRITC (5/6-tetramethyl-rhodamine isothiocyanate), mixed isomer Thermo Scientific™

Thermo Scientific TRITC is a high-performance derivative of rhodamine dye, activated for easy and reliable labeling of antibodies, proteins and other molecules for use as fluorescent probes.

Features of TRITC:

Amine-specific labeling—TRITC varieties of rhodamine efficiently label antibodies and other purified proteins at primary amines (lysine side chains)
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios for optimum activity and fluorescence

Tetramethylrhodamine isothiocyanate (TRITC) is an amine-reactive derivative of rhodamine dye that has wide-ranging application as antibody and other probe labels for use in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as western blotting and ELISA.

Applications:
• Label antibodies for use as immunofluorescent probes
• Label oligonucleotides for hybridization probes
• Detect proteins in gels and on western blots

Properties of Rhodamine Dyes:
Thermo Scientific Pierce Rhodamine Dyes are mixtures of isomers with reactive groups attached at the 5- and 6-positions of the bottom ring. The properties of these isomers are indistinguishable in terms of excitation and emission spectra, and for protein applications there is no need to isolate a specific isomer.

TRITC is the base tetramethylrhodamine molecule functionalized with an isothiocyanate reactive group (—N=C=S) at one of two hydrogen atoms on the bottom ring of the structure. This derivative is reactive towards primary amine groups on proteins, peptides and other biomolecules.

Application Data:

Related Products
Pierce™ NHS-Rhodamine Antibody Labeling Kit
NHS-Rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester), mixed isomer

Click-iT™ SDP Ester sDIBO Alkyne Invitrogen™

Click-iT SDP Ester sDIBO Alkyne reacts with azides via a copper-free Click chemistry reaction to produce SDP Ester (amine-reactive) bioconjugates. sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less “sticky” and give lower signal background in biological samples. Copper-free Click bio-conjugation reactions are ideal for surface labeling of live cells and also minimize damage to enzymes and fluorescent proteins like GFP or R-PE. Macromolecules that have been azide-modified enzymatically, chemically, or metabolically can be now be labeled easily, yielding more soluble bioconjugates with improved biological labeling utility.

• More soluble than DIBO cyclooctynes leading to more soluble conjugates
• Minimal background potential in cells and tissues compared to original DIBO cycloctynes

D-Biotin Invitrogen™

D-biotin binds with high affinity to streptavidin, avidin and NeutrAvidin® biotin-binding proteins.

CellTracker™ Green CMFDA Dye Invitrogen™

CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate) is a fluorescent dye well suited for monitoring cell movement or location. After loading into cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The green excitation/emission spectra are ideal for multiplexing with 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)
• Green excitation/emission spectra (492/517 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

CellTracker™ Green CMFDA fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into a cell-impermeant, fluorescent product. CellTracker™ Green fluorescent dyes are retained in living cells through several generations. The dyes are transferred to daughter cells but not adjacent cells in a population. CellTracker™ Green CMFDA 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™ Green CMFDA dye are well separated from RFP (red fluorescent protein) spectra allowing for multiplexing.

CellTrace™ Calcein Violet, AM, for 405 nm excitation - Special Packaging Invitrogen™

CellTrace™ calcein violet AM is an optimal dye for distinguishing live cells through the presence of ubiquitous intracellular esterase activity. The enzymatic conversion of the virtually non-fluorescent cell-permeant calcein violet AM to the intensely fluorescent calcein violet (ex/em 400/452 nm) can be easily detected with the violet laser, allowing other laser lines to be used for conventional fluorochromes.

View a selection guide for all Nonfixable Viability Dyes for Flow Cytometry.

DyLight™ 650-Phosphine Thermo Scientific™

Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.

When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

General features of the phosphine-activated DyLight Fluors:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that fluorescent labeling is specific
High-performance fluorescence—DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)

Related Products
DyLight™ 488-Phosphine
DyLight™ 550-Phosphine

DyLight™ 650 Antibody Labeling Kit Thermo Scientific™

The Thermo Scientific DyLight 650 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 650 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection 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 amine-reactive DyLight 650 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 650 provides vibrant far-red fluorescence with comparable or improved performance over other dyes, including Alexa Fluor™ 647 and Cy5™ dye, for fluorescent 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 650 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 650 NHS Ester:

High performance—DyLight 650 fluoresces brighter than Alexa Fluor 647 and Cy5
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 650 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 650 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

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DyLight™ 650 NHS Ester
DyLight™ 650 Microscale Antibody Labeling Kit

Alexa Fluor™ 790 NHS Ester (Succinimidyl Ester) Invitrogen™

Alexa Fluor® 790 is a bright and photostable near-IR dye that is spectrally similar to indocyanine green (ICG) and the IRDye® 800 dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 790 dye is water soluble and pH-insensitive from pH 4 to pH 10. Fluorescence of this long-wavelength Alexa Fluor® dye is not visible to the human eye but is readily detected by most imaging systems. As the longest-wavelength Alexa Fluor® dye, the emission is well separated from commonly used far-red fluorophores such as Alexa Fluor® 647 dye or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also expected to be useful for small animal in-vivo imaging (SAIVI) applications or for two-color western applications with the LI-COR® Odyssey® infrared imaging system. In addition to reactive dye formulations, we offer Alexa Fluor® 790 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 Alexa Fluor® 790 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® 790 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 784/814 nm
Extinction coefficient: 260,000 cm-1M-1
Spectrally similar dyes: indocyanine green (ICG) and the IRDye® 800 dye
Molecular weight: ~1750

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.

Click-iT™ Alexa Fluor™ 488 sDIBO Alkyne for Antibody Labeling Invitrogen™

The Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling is optimized for easy attachment to azido modified antibodies using copper-free Click chemistry. This sDIBO label can be used with antibodies that have been modified using the SiteClick Antibody Azido Modification Kit or antibodies that have been engineered to contain azido moieties. These sDIBO alkynes are improved versions of our original DIBO cyclooctynes, yielding conjugates that are less 'sticky' and give lower signal background in biological samples.

This modular labeling system gives you the option to choose different fluorescent labels for your antibody and attach another molecule via streptavadin or your own molecule via amine-reactive or amine-containing moieties depending on your assay.

There are multiple Click-iT sDIBO labels to choose from:
Click-iT Alexa Fluor 488 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 555 sDIBO Alkyne for Antibody Labeling
Click-iT Alexa Fluor 647 sDIBO Alkyne for Antibody Labeling
Click-iT Biotin sDIBO Alkyne for Antibody Labeling
Click-iT Amine sDIBO Alkyne for Antibody Labeling
Click-iT SDP Ester sDIBO Alkyne for Antibody Labeling

Learn more about SiteClick labeling technology ›

Custom SiteClick Antibody Labeling Service and sDIBO labels
If you have an antibody that is considered 'difficult to label' or has lost activity after labeling using a conventional method, please contact our custom service representatives to determine whether the SiteClick Antibody Labeling Service would be right for your antibody. We offer complete custom SiteClick antibody labeling services with the option of multiple detection molecules including biotin, Alexa Fluor dyes, Qdot fluorophores, R-PE, chelates for PET imaging, and many others.

Biotin-X Cadaverine (5-(((N-(Biotinoyl)Amino)Hexanoyl)Amino)Pentylamine, Trifluoroacetic Acid Salt) Invitrogen™

The primary aliphatic amine of biotin-X cadaverine can be reversibly coupled to aldehydes and ketones to form a Schiff base - which can be reduced to a stable amine derivative by sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNH3) to form new biotinylated probes. Carboxylic acids of proteins and other water-soluble biopolymers can be coupled to this molecule in aqueous solution using water-soluble carbodiimides such as EDAC (E2247).

Alexa Fluor™ 568 NHS Ester (Succinimidyl Ester) Invitrogen™

Alexa Fluor® 568 is a bright orange dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 568 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 568 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® 568 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® 568 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 578/602 nm
Extinction coefficient: 88,000 cm-1M-1
Spectrally similar dyes: Rhodamine Red
Molecular weight: 791.8

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.

Alexa Fluor™ 488 C5 Maleimide Invitrogen™

Alexa Fluor® 488 is a bright, green fluorescent dye with excitation ideally suited to the 488 nm 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).

The maleimide derivative of Alexa Fluor® 488 is the most popular tool for conjugating the dye to a thiol group on a protein, oligonucleotide thiophosphate, or low molecular weight ligand. The resulting Alexa Fluor® 488 conjugates exhibit brighter fluorescence and greater photostability than the conjugates of other spectrally similar fluorophores.

Detailed information about this AlexaFluor® maleimide:

Fluorophore label: Alexa Fluor® 488 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 493/516 nm
Extinction coefficient: 72,000 cm-1M-1
Spectrally similar dyes: Fluorescein (FITC), Cy®2
Molecular weight: 720.66

Typical Conjugation Reaction
The protein should be dissolved at a concentration of 50-100 µM in a suitable buffer (10-100 mM phosphate, Tris, or HEPES) at pH 7.0-7.5. In this pH range, the protein thiol groups are sufficiently nucleophilic that they react almost exclusively with the reagent in the presence of the more numerous protein amine groups, which are protonated and relatively unreactive. We recommend reducing any disulfide bonds at this point using a 10-fold molar excess of reducing agent such as DTT or TCEP. Excess DTT must be removed by dialysis and subsequent thiol-modification should be carried out under oxygen-free conditions to prevent reformation of the disulfide bonds; these precautions are not necessary when using TCEP prior to maleimide conjugation.

The Alexa Fluor® maleimide is typically dissolved in high-quality anhydrous dimethylsulfoxide (DMSO) at a concentration of 1-10 mM immediately prior to use, and stock solutions should be protected from light as much as possible. Generally, this stock solution is added to the protein solution dropwise while stirring to produce approximately 10-20 moles of reagent per mole of protein, and the reaction is allowed to proceed at room temperature for 2 hours or at 4°C overnight, protected from light. Any unreacted thiol-reactive reagent can be consumed by adding excess glutathione, mercaptoethanol, or other soluble low molecular weight thiol.

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.
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