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Qdot™ 525 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 525 ITK™ amino (PEG) quantum dots are the ideal starting material for preparing custom conjugates of ultrabright and photostable fluorescently labeled proteins or other biopolymers. These probes are functionalized with amine-derivatized PEG, which prevents non-specific interactions and provides a convenient handle for conjugation. The amino quantum dots react efficiently with isothiocyanates and succinimidyl esters, or with native carboxylic acids using water-soluble carbodiimides such as EDC. Such derivatives may be used for various labeling and tracking applications that require ultrabright and stable fluorescence. Our Qdot® ITK™ amino quantum dots are provided as 8 µM solutions and are available in 8 colors of Qdot® probes.

Important Features of Qdot® ITK™ Amino Quantum Dots:
• Qdot® 525 ITK™ amino quantum dot has emission maxima of ~525 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large stokes shift
• Available in multiple colors
• Ideal for various labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the amine-derivatized form, we offer Qdot® ITK™ quantum dots with carboxyl and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Qdot™ 585 ITK™ Amino (PEG) Quantum Dots (Invitrogen™)

Qdot® 585 ITK™ amino (PEG) quantum dots are the ideal starting material for preparing custom conjugates of ultrabright and photostable fluorescently labeled proteins or other biopolymers. These probes are functionalized with amine-derivatized PEG, which prevents non-specific interactions and provides a convenient handle for conjugation. The amino quantum dots react efficiently with isothiocyanates and succinimidyl esters, or with native carboxylic acids using water-soluble carbodiimides such as EDC. Such derivatives may be used for various labeling and tracking applications that require ultrabright and stable fluorescence. Our Qdot® ITK™ amino quantum dots are provided as 8 µM solutions and are available in 8 colors of Qdot® probes.

Important Features of Qdot® ITK™ Amino Quantum Dots:
• Qdot® 585 ITK™ amino quantum dot has emission maxima of ~585 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large stokes shift
• Available in multiple colors
• Ideal for various labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the amine-derivatized form, we offer Qdot® ITK™ quantum dots with carboxyl and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

5-(Aminomethyl)Fluorescein, Hydrochloride (Invitrogen™)

The primary aliphatic amine of 5-(aminomethyl)fluorescein 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 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).

CellVue™ Maroon Cell Labeling Kit (Invitrogen™)

CellVue™ dyes are lipophilic dyes that can be used to label the cell membrane for the purpose of identifying and tracking labeled cells. Cell labeling is rapid and stable and can be combined with fluorescently labeled antibodies and other markers of cellular function for flow cytometric analysis and fluorescent microscopy. The Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C).

CellVue™ Maroon is a far-red fluorescent cell labeling reagent. It is optimally excited at 647 nm and has a peak emission of 667 nm; however, it can be detected equally in filter sets designed for APC, Alexa Fluor™ 700, and APC-eFluor™ 780 or similar fluorochromes. Therefore, using antibodies conjugated to these fluorochromes in combination with using CellVue™ Maroon is not recommended.

Reported Application
Microscopy, Cell Labeling, Immunocytochemistry, Flow Cytometric Analysis

3-Amino-3-Deoxydigoxigenin Hemisuccinamide, Succinimidyl Ester (Invitrogen™)

The amine-reactive version of digoxigenin (3-amino-3-deoxygigoxigenin hemisuccinamide, SE) can be used to attach this important hapten to biomolecules of interest for subsequent detection with an anti-digoxigenin antibody.

Oregon Green™ 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer (Invitrogen™)

The amine-reactive Oregon Green® 488 carboxylic acid, succinimidyl ester can be used to can be used to create green fluorescent bioconjugates with excitation/emission maxima ~496/524 nm. This fluorinated analog of fluorescein overcomes some of the key limitations of fluorescein, including greater photostability and a lower pKa (pKa ~ 4.7 versus 6.4 for fluorescein), making its fluorescence essentially pH insensitive in the physiological pH range.

Eosin-5-Maleimide (Invitrogen™)

Bioconjugates made with the thiol-reactive eosin-5-maleimide can be used as phosphorescent probes or as photosensitizers. With its high quantum yield (~0.57) for singlet oxygen generation, eosin and its conjugates can be used as effective photooxidizers of diaminobenzidine (DAB) in high-resolution electron microscopy studies and in correlated fluorescence and electron microscopy applications.

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).

Texas Red™-X, Succinimidyl Ester, mixed isomers (Invitrogen™)

The isomeric mixture of amine-reactive Texas Red-X, succinimidyl ester and its conjugates emit at a longer wavelength than do either tetramethylrhodamine or Lissamine rhodamine B, and are among the most commonly used long-wavelength "third labels" in fluorescence microscopy and flow cytometry. Protein conjugates prepared with Texas Red-X succinimidyl ester also have a higher fluorescence yield than those with the same labeling ratio prepared with Texas Red sulfonyl chloride.

Qtracker™ 705 Vascular Labels (Invitrogen™)

Qtracker® non-targeted quantum dots are designed to be injected into the tail vein of mice for the study of vascular structure using small animal in vivo imaging (SAIVI) techniques. These nanocrystals exhibit intense fluorescence with red-shifted emission for increased tissue penetration, and have a PEG surface coating specially developed to minimize nonspecific interactions and reduce immune response by the tissue. Because the PEG surface coating does not contain reactive functional groups, the Qtracker® non-targeted quantum dots are retained in circulation longer and can be imaged for up to 3 hours with a single injection or for longer periods of time with additional injections.

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

Key Attributes:

Qtracker® 705 label has Ex/Em (405-665/705) nm
Designed for small animal in vivo imaging
Introduced via tail vein injection, can be imaged for up to 3 hours after injection
Available in four colors—565 nm, 655 nm, 705 nm, or 800 nm emission

Read more about SAIVI and about applications for Qdot® nanocrystals.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ CFSE Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Non-toxic—staining does not adversely effect cell health
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ CFSE dye enables the visualization of eight or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ CFSE dye easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye is designed to provide a consistent signal, even after several days in a cell culture environment.

Non-toxic
CellTrace™ CFSE dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells. Researchers have used CFSE SE labeling to show that transplantable hematopoietic cells proliferate in vitro in response to stimulation by a growth factor cocktail.

Simple, robust staining protocol
The CellTrace™ CFSE Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

DACM, N-(7-Dimethylamino-4-Methylcoumarin-3-yl))Maleimide (Invitrogen™)

The thiol reactive coumarin, DACM can be used to create blue-fluorescent bioconjugates. When compared with AMCA conjugates, conjugates of the UV light-excitable 7-dialkylaminocoumarin fluorophore have slightly longer-wavelength emission spectra (~470 nm). The unreacted reagent is nonfluorescent and can also be used to quantitate free thiols.

APEX™ Alexa Fluor™ 594 Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


Better Results and Workflows With Primary Labeled Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

EZ-Link™ NHS-PEG4 Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link NHS-PEG4 Biotinylation Kit contains EZ-Link NHS-PEG4-Biotin and accessory reagents required for 8 1–10 mg protein labeling reactions. EZ-Link NHS-PEG4-Biotin is a pegylated, water-soluble reagent for simple and efficient biotin labeling of antibodies, proteins and other primary amine-containing macromolecules.

Features of EZ-Link NHS-PEG4-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Pegylated – spacer arm contains a hydrophilic, 4-unit, polyethylene glycol (PEG) group
Enhances solubility – pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Long reach – spacer arm (total length added to target) is 29 angstroms; this reduces steric hindrance when binding to avidin molecules

NHS-PEG4-Biotin is a long (29.0Å), pegylated, water-soluble, NHS-ester biotinylation reagent to label amines and maximize solubility of antibodies and other proteins. The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups to form stable amide bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives the reagent a long and flexible connection to minimize steric hindrance for binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

Related Products
EZ-Link™ NHS-PEG4-Biotin
EZ-Link™ Micro NHS-PEG4-Biotinylation Kit

DyLight™ 633 Antibody Labeling Kit (Thermo Scientific™)

The Thermo Scientific DyLight 633 Antibody Labeling Kit contains an NHS ester-activated derivative of high-performance DyLight 633 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 633 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 633 fluoresces red and has physical properties comparable to other 633 dyes, including Alexa Fluor™ 633. 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 633 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 633 NHS Ester:

High performance—DyLight 633 shows brighter fluorescence than Alexa Fluor 633
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 633 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 DyLight 633 Amine-Reactive 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 633 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

Related Products
DyLight™ 633 NHS Ester
DyLight™ 633 Microscale Antibody Labeling Kit

Alexa Fluor™ 555 C2 Maleimide (Invitrogen™)

Alexa Fluor® 555 is a bright orange dye. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 555 dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 555 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® 555 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® 555 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® 555 dye
Reactive group: maleimide
Reactivity: thiol groups on proteins and ligands, oligonucleotide thiophosphates
Ex/Em of the conjugate: 556/572 nm
Extinction coefficient: 158,000 cm-1M-1
Spectrally similar dyes: Tetramethylrhodamine
Molecular weight: ~1250

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.

EZ-Link™ Biocytin (Thermo Scientific™)

Thermo Scientific EZ-Link Biocytin is a simple variant of biotin that contains a primary amine in its valeric acid chain, which provides the essential backbone for construction of certain long-chain and trifunctional biotinylation reagents.

Features of EZ-Link Biocytin:

Biotinylation—label molecules and surfaces for assays or affinity purification methods involving avidin or streptavidin probes and resins
Amine-activated—primary amine can be crosslinked to proteins and material surfaces using EDC and other crosslinkers
Lysine derivative—functions as a biotin-tagged amino acid; potentially useful as a control or quenching reagent in biotin assay methods
Medium length—spacer arm (total length added to target) is 20.1 angstroms, representing a 7-atom extension of the native biotin valeric acid

Biocytin is ε-N-[d-biotinyl]-L-lysine, a compound formed by conjugation of the epsilon amine of lysine to the valeric acid side chain of biotin. It contains terminal carboxyl and amino groups, which provide functional handles for derivatization or conjugation to proteins, surfaces and other molecules. Carbodiimide (EDC) and NHS-ester crosslinker chemistries are most often utilized for covalent modifications involving biocytin. The compound is also useful as an amino acid control or biotin standard in assay methods involving streptavidin binding.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Amino-biotin compounds can be conjugated to functional groups of proteins and other molecules in a variety of ways. The most common method is to crosslink the terminal primary amine to carboxyl groups using . Carboxyl groups (-COOH) occur in aspartate or glutamate residues and the carboxy-terminus of polypeptides. When activated with EDC (Part No. 22980), carboxylates react with amino (—NH2) groups to form amide bonds.

Biocytin has both carboxyl and amino groups. Therefore, to prevent self-conjugation of biocytin, EDC-mediated reaction schemes with this compound are usually done in two steps, aided by Sulfo-NHS (Part No. 24510): (1) Activate a carboxylate molecule using EDC and Sulfo-NHS, followed by complete removal or inactivation of the EDC reagent; then (2) Add Biocytin to allow its primary amine to react with the Sulfo-NHS ester-activated carboxyl groups. See NHS-ester Chemistry. Subsequently, the remaining carboxyl group of biocytin could be conjugated to yet another molecule. For example, biocytin is the starting material for synthesis of Sulfo-SBED (Part No. 33033), a trifunctional reagent.

pHrodo™ Red Maleimide (Invitrogen™)

The thiol-reactive pH-sensitive pHrodo® Red Maleimide dye is suitable for the creation of bioconjugates to study endocytosis and phagocytosis. pHrodo® Red dramatically increases fluorescence as the pH of its surroundings become more acidic.
• Use pH-sensitive pHrodo® Red Maleimide to make pH-sensitive bioconjugates of your choice
• Get faster, more accurate results than with any other endocytosis or phagocytosis assay—no need for wash steps or quenchers
• Multiplex with green fluorescent markers such as GFP, pHrodo® Green, and many others

The increase in fluorescence of pHrodo® Red as pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it an ideal tool to study endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo® Red makes it useful for multi-color experiments. pHrodo® Red has been validated for use on a variety of platforms including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo® Red in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo® Red Maleimide is a thiol-reactive dye that can be used to create pHrodo® Red bioconjugates in aqueous buffer. The maleimide reacts with free sulphydryl groups produced by the reduction of cysteines in proteins or peptides. Maleimides are particularly useful for labeling antibodies as the dye will not attach to the antibody binding site. This reaction will result in a stable conjugate that can be used in live cell assays or stored for later use.

pHrodo® Red is also available in an amine-reactive form (see pHrodo® Red SE), as well as a selection of ready-to-use conjugates (e.g., E. coli, S. aureus, and dextran). In addition, pHrodo® Green reactive dyes and ready-to-use conjugates are available as a color alternative with the same properties.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

DyLight™ 650 Maleimide (Thermo Scientific™)

Thermo Scientific DyLight 650 Sulfhydryl-Reactive Dye is a maleimide-activated derivative of high-performance DyLight 650 used to fluorescently label sulfhydryl-containing peptides, proteins, and other biomolecular probes.

DyLight 650 provides vibrant far-red fluorescence with comparable or improved performance over other dyes, including Alexa Fluor™ 647 and Cy5™ dye, over a broad pH range (pH 4-9). The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates.

Features of DyLight 650 Maleimide:

High performance—DyLight 650 fluoresces brighter than Alexa Fluor 647 and Cy5
Specific—maleimide-activated dye labels proteins and other molecules at reduced sulfhydryls (-SH)
Efficient labeling methods—well-characterized chemistry and optimized protocols provide for reliable, high-quality labeling
Optimized antibody labeling procedure—complete protocol for IgG reduction and labeling and calculating the labeling efficiency

Applications:
• Antibody labeling for immunofluorescence applications, including immunocytochemistry (ICC), immunohistochemistry (IHC), Western blotting and ELISA assay
• Target macromolecule labeling for in vitro and in vivo fluorescent detection strategies

DyLight 650 Sulfhydryl-Reactive Dye is activated with a maleic acid imide (maleimide) moiety to form a reactive alkylation reagent. Labeling occurs through reaction of the maleimide-activated dye with reduced sulfhydryl groups (-SH) to form stable thioether bonds. Maleimides are specific for sulfhydryl groups between pH 6.5–7.5. Learn more about maleimide chemistry.

Zenon™ Alexa Fluor™ 546 Mouse IgG1 Labeling Kit (Invitrogen™)

Zenon® labeling technology provides a fast, versatile, and reliable method for adding a fluorescent label to an antibody. You need only a small amount of starting material, and the method is optimized for efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Antibody conjugates formed using Zenon® technology may be used in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, and high-throughput applications. This exclusive Molecular Probes® Zenon® labeling technology greatly simplifies the use of multiple mouse-derived antibodies in the same staining protocol.

Important Features of Zenon® Labeling Technology:

• Labeled antibodies typically ready to use in 10 minutes
• Requires only 1–20 μg primary antibody
• Simple, no purification required
• Flexible–over 24 fluorophores plus biotin, HRP, alkaline phosphatase, and TSA to choose from
• Multiplex with other mouse monoclonal antibodies simultaneously


Save Time and Antibody
Each kit comes with affinity-purified monovalent Fab fragment of a goat anti-Fc antibody (or, in the case of the Zenon® Goat IgG Labeling Kits, a rabbit anti-Fc antibody) that has been conjugated to one of our premier Alexa Fluor® dyes or to Pacific Blue™, Pacific Orange™, fluorescein, or Texas Red®-X dyes, biotin R-phycoerythrin (R-PE), allophycocyanin (APC), HRP, or alkaline phosphatase.

Formation of the Fab–antibody complex with the Zenon® Antibody Labeling Kits is extremely fast (5 min for complex, 5 min for blocking step). And Zenon® labeling is a reliable and reproducible method, even with as low 0.4 μg in 2 μL of primary antibody. There is minimal waste of expensive or difficult-to-obtain antibodies when using the Zenon® Antibody Labeling Kits.

Preserve Primary Antibody Function and Affinities
Reactive dye labeling of primary antibodies can have unpredictable and undesirable outcomes. Among these are reduced binding affinities by label addition in the binding pocket. Zenon® antibody labeling approach, targeted to the Fc tail, avoids this concern.

Moreover the Zenon® dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to help ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. The procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.

Many Fluorophore and Enzyme Labels Available
Zenon® immunolabeling technology makes it very easy to change fluorescent color combinations or detection methodologies by simply using a different dye- or enzyme-labeled Fab fragment from our extensive selection of over 100 Zenon® Antibody Labeling Kits. If larger quantities or covalent attachment of the label is desired, see Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices.

Zenon® Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol
The stability of the Zenon® complex is sufficient to allow sequential (or simultaneous) labeling of different targets in cells and tissues with multiple antibody complexes. Subsequent to staining, an aldehyde-based fixation step can permanently block the transfer of Zenon® labels between different primary antibodies and will preserve the staining pattern.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Related Links:

Zenon® Labeling Technology
Zenon® Technology: Versatile Reagents for Immunolabeling—Section 7.3

CellVue™ Lavender Cell Labeling Kit (Invitrogen™)

CellVue™ dyes are lipophilic dyes that can be used to label the cell membrane for the purpose of identifying and tracking labeled cells. Cell labeling is rapid and stable and can be combined with fluorescently labeled antibodies and other markers of cellular function for flow cytometric analysis and fluorescent microscopy. Mini CellVue™ Kits are supplied with one vial of dye stock (1 mM in ethanol) and one vial of labeling vehicle (Diluent C).

CellVue™ Lavender is a violet fluorescent cell labeling reagent. It is optimally excited at 420 nm and has a peak emission of 461 nm. CellVue™ Lavender is compatible with most multi-color flow cytometric applications; however, due to similar emission properties it cannot be used in combination with eFluor™ 450 or Pacific Blue™ reagents.

Reported Application
Flow Cytometric Analysis, Microscopy, Immunocytochemistry, Cell Labeling

Badan (6-Bromoacetyl-2-Dimethylaminonaphthalene) (Invitrogen™)

The thiol reactive badan generally reacts with thiols more slowly than iodoacetamides or maleimides, but does form very strong thioether bonds that are expected to remain stable under conditions required for complete amino acid analysis. The fluorescence emission peak and intensity of these adducts are particularly sensitive to conformational changes or ligand binding, making this dye one of the most useful thiol-reactive probe for protein structure studies. The environment-sensitive spectral shift of badan conjugates may make this useful for distinguishing thiols that are located in membranes versus those exposed to aqueous solvation in cells.

CellTracker™ Violet BMQC Dye (Invitrogen™)

CellTracker™ Violet BMQC 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 violet 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)
• Violet excitation/emission spectra (415/516 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

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

Alexa Fluor™ 594 Microscale Protein Labeling Kit (Invitrogen™)

Microscale Protein Labeling Kits provide a convenient means for attaching a fluorescent label to a small amount of antibody or protein (20–100 µg). The kits are available in four Alexa Fluor® colors (or biotin) and supply everything needed for three labeling and separation reactions.

Important Features of Microscale Protein Labeling Kits:
• Labeled proteins typically ready to use typically in 2 hours (~30 minutes hands-on time)
• Optimized for 20–100 µg of protein with molecular weights between 12 and 150 kDa
• Purified using convenient spin filters with yields between 60 and 90%
• Stabilizing proteins must be removed from the sample before labeling

Stable Reaction Chemistry and Superior Alexa Fluor® Dyes
In the Microscale Protein Labeling Kits, the reactive dye contains a succinimidyl (NHS) ester moiety that reacts with primary amines of proteins to form stable dye-protein conjugates. Compared to traditional dyes, Alexa Fluor® dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor® dyes, higher degrees of labeling can be achieved without intramolecular quenching. For details see Alexa Fluor® Dyes Spanning the Visible and Infrared Spectrum—Section 1.3.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

SiteClick™ Qdot™ 605 Antibody Labeling Kit (Invitrogen™)

Create a perfectly labeled antibody with the SiteClick™ Qdot® 605 Antibody Labeling Kit. This kit replaces the conventional Qdot® 605 Antibody Conjugation Kit (Q22001MP). Unlike the conventional amine-thiol crosslinker method, SiteClick™ labeling specifically attaches the label to the heavy chains of an IgG antibody ensuring that the antigen binding domains remain available for binding to your antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. In addition, no harsh reduction steps are required, and the labeling is consistent and reproducible each time it is performed. Depending upon the label, the resulting SiteClick™ labeled antibody can be used in flow cytometry, fluorescence imaging, or western blot detection.

Important Features of the SiteClick™ Qdot® 605 Antibody Labeling Kit:

• Contains everything required to label 100 µg of IgG antibody
• Easy to follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate.
• Qdot® 605 labels can be used in confocal or traditional fluoresce microscopy.
• In flow cytometry, Qdot® 605 can be excited by the 488 nm line of the argon-ion laser, or alternatively via excitation at 405 nm. This is true of all Qdot® fluorophores.

Qdot® Fluorophores Are Our Brightest Labels
Antibody conjugates made with Qdot® fluorophores produce fluorescence output that surpasses that of traditional organic dyes. Paired with the correct optical filters, Qdot® nanocrystals are as much as 50 times brighter. Read more about Qdot® nanocrystals or review additional product details in Qdot® Nanocrystals—Section 6.6 in the Molecular Probes® Handbook.

Learn More About Protein and Antibody Labeling
We offer a wide selection of Molecular Probes® antibody and protein labeling kits (see Antibody Labeling from A to Z). To learn more about our various kits, read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

Custom SiteClick™ Antibody Labeling Services
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, and others.

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

Zenon™ Allophycocyanin Rabbit IgG Labeling Kit (Invitrogen™)

Zenon labeling technology provides a fast, versatile and reliable method for producing antibody conjugates, even with very small (submicrogram) amounts of starting material. Antibody conjugates formed using Zenon technology may be used to stain cells in any protocol where a directly labeled primary antibody is suitable, including flow cytometry, imaging, high throughput and other applications. Moreover, this technology simplifies applications that previously were time consuming or not practical, such as the use of multiple mouse-derived antibodies in the same staining protocol.

View a selection guide for all Zenon™ antibody labeling kits and other antibody labeling products.

Click-IT™ Fucose Alkyne (Invitrogen™)

Identify and characterize fucosylated proteins with Click-iT® fucose alkyne using the powerful click chemistry, a simple and robust two-step labeling and detection technique. In step one, the alkyne-containing biomolecule is fed to cells or animals and actively incorporated into proteins. Unlike other labels such as biotin or a fluorescent dye, the alkyne-tag is small enough that the tagged molecule is an acceptable substrate for the enzymes that incorporate this building block into proteins. Detection utilizes the chemoselective ligation or “click" reaction between an azide and an alkyne where the modified protein is detected with the corresponding azide-containing dye or hapten using either the Click-iT® Cell Reaction Buffer Kit or the Click-iT® Protein Buffer Kit. With the Click-iT® Cell Reaction Buffer Kit, cells can be analyzed by fluorescence microscopy, flow cytometry or high-content imaging and analysis (HCS) together with other biomarkers of interest for content and context rich results. With the Click-iT® Protein Reaction Buffer Kit, achieve detection sensitivity in 1-D gels and western blots in the low femtomole range or perform LC-MS⁄MS and MALDI MS analysis.

ManNAz (N-azidoacetylmannosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce ManNAz (N-azidoacetylmannosamine-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
GlcNAz (N-azidoacetylglucosamine tetraacylated)
GalNAz (N-azidoacetylgalactosamine tetraacylated)

Click-IT™ Alexa Fluor™ 594 DIBO Alkyne, for copper free click chemistry detection of azide (Invitrogen™)

The fluorescent Alexa Fluor® 594 Dibo alkyne is reactive with azides via a copper-free "click chemistry" reaction.

Please consider this copper-free variation to our copper requiring alkynes if you are staining the surface of live cells or have concerns about native protein function loss with copper in cell extracts. Copper can damage fluorescent proteins, Quantum Dot nanocyrstals, certain enzymes and photoproteins like RPE. The DIBO reagent is not suitable for staining intracellular components of fixed and permeabilized cells due to high backgrounds.

Alexa Fluor™ 488 Hydroxylamine (Invitrogen™)

Alexa Fluor® 488 Hydroxlamine 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® hydroxlamine:

• Fluorophore label : Alexa Fluor® 488 dye
• Reactive group: hydroxlamine
• Reactivity: Aldehydes or ketones
• Ex/Em of the conjugate: 494/518 nm
• Extinction coefficient: 77,000 cm-1M-1
• Spectrally similar dyes: FITC, GFP
• Molecular weight: 895.07

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)

Alexa Fluor™ 610-X NHS Ester (Succinimidyl Ester) (Invitrogen™)

Alexa Fluor® 610-X is a bright and photostable Texas Red® dye substitute. Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 610-X dye is water soluble and pH-insensitive from pH 4 to pH 10. In addition to reactive dye formulations, we offer Alexa Fluor® 610–R-Phycoerythrin tandem antibody conjugates for flow cytometry.The NHS ester (or succinimidyl ester) of Alexa Fluor® 610-X 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® 610-X dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 603/623 nm
Extinction coefficient: 144,000 cm-1M-1
Spectrally similar dyes: Texas Red®
Molecular weight: 1284.8

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.

6-ROX, SE (6-Carboxy-X-Rhodamine, Succinimidyl Ester), single isomer (Invitrogen™)

The isomeric 6-ROX, SE is an amine-reactive form of carboxy-X-rhodamine and is widely used for oligonucleotide labeling and automated DNA sequencingapplications.

Alexa Fluor™ 647 Protein Labeling Kit (Invitrogen™)

Molecular Probes® Protein Labeling Kits provide a convenient means for attaching a fluorescent label (or biotin) to an antibody (or a protein larger than 40 kDa). Conjugates are ideal for multiple applications, including flow cytometry, fluorescent microscopy, immunohistochemistry, primary detection, ELISAs, immunocytochemistry, FISH, and more. Kits are available in 12 Alexa Fluor® dye colors, biotin, the hapten Oregon Green® 488, fluorescein EX, and Texas Red® dye. Each kit provides the components needed to perform three protein conjugations and purifications.

Important Features of Protein Labeling Kits:

• Labeled proteins typically ready to use in 2 hr (~30 min hands-on time)
• Designed to label 1 mg of IgG
• Simple protocol—react, separate, use
• Stabilizing proteins must be removed from the sample before labeling


The Benefits of Alexa Fluor® Dyes
Compared to traditional dyes, Alexa Fluor® dyes are brighter, more photostable, and more pH resistant between pH 4 and 10. And generally when using Alexa Fluor® dyes, higher degrees of labeling can be achieved without intramolecular quenching. For details see Alexa Fluor® Dyes Spanning the Visible and Infrared Spectrum—Section 1.3.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

5-SFX (6-(Fluorescein-5-Carboxamido) Hexanoic Acid, Succinimidyl Ester), single isomer (Invitrogen™)

Searching for superior alternatives to fluorescein? Our Alexa Fluor Dye Series offers everything you're looking for and more.

CellTracker™ Orange CMTMR Dye (Invitrogen™)

CellTracker™ Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (mixed isomers) 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 orange excitation/emission spectra are ideal for multiplexing with green 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)
• Orange excitation/emission spectra (541/565 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

CellTracker™ Orange CMTMR fluorescent dye has been designed to freely pass through cell membranes into cells, where it is transformed into cell membrane-impermeant reaction products. CellTracker™ Orange CMTMR dye is retained in living cells through several generations. The dye is transferred to daughter cells, but not adjacent cells in a population. CellTracker™ Orange CMTMR 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™ Orange CMTMR dye are well separated from GFP (green fluorescent protein) and spectra allowing for multiplexing.

DyLight™ 800-4xPEG NHS Ester (Thermo Scientific™)

Thermo Scientific DyLight 800-4xPEG Amine-Reactive Dye is a derivative of our high-performance DyLight 800 Dye that can be used to fluorescently label antibodies and other proteins for near-IR detection.

The DyLight 800-4xPEG 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 800-4xPEG Dye can be used as molecular probes for cellular imaging and other fluorescence detection methods. The NIR fluorescence properties of DyLight 800-4xPEG 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 800-4xPEG NHS Ester:

High fluorescence intensity—fluorescence comparable to Alexa Fluor™ 800 and IRDye™ 800
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 800-4xPEG 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.

Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)-TRITC), mixed isomers (Invitrogen™)

The thiol-reactive tetramethylrhodamine-5-(and-6)-isothiocyanate (5(6)-TRITC) can be used to can be used to create bright orange-red-fluorescent bioconjugates with excitation/emission maxima ~555/580.

EZ-Link™ Micro Sulfo-NHS-Biotinylation Kit (Thermo Scientific™)

The Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit contains reagents sufficient for 8 biotinylation reactions (e.g., 0.05–0.2 mg antibody per reaction). The EZ-Link Sulfo-NHS-Biotin reagent included in the kit is a short-chain, water-soluble biotinylation reagent used for labeling antibodies, proteins and other molecules that have primary amines.

Features of EZ-Link Sulfo-NHS-Biotin:

Protein labeling—biotinylate antibodies to facilitate immobilization, purification or detection using streptavidin resins or probes
Cell surface labeling—biotinylates only surface proteins of whole cells because the negatively charged reagent does not permeate cell membranes
Amine-reactive—reacts with primary amines (-NH2), such as lysine side-chains or the amino-termini of polypeptides
Soluble—charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
Very short—spacer arm (total length added to target) is 13.5 angstroms; it consists of the native biotin valeric acid group only.

Sulfo-NHS-Biotin is the shortest of three very similar EZ-Link Reagents that are water-soluble, non-cleavable, and enable simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing macromolecules in solution. Specific labeling of cell surface proteins is another common application for these uniquely water-soluble and membrane impermeable reagents. Differing only in their spacer arm lengths, the three Sulfo-NHS-ester reagents offer the possibility of optimizing labeling and detection experiments where steric hindrance of biotin binding is an important factor. Sulfo-NHS-Biotin is offered in several package sizes and as complete protein labeling kits.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

Related Products
EZ-Link™ Sulfo-NHS-Biotin
EZ-Link™ Sulfo-NHS-Biotinylation Kit

APEX™ Alexa Fluor™ 568 Antibody Labeling Kit (Invitrogen™)

The APEX® Antibody Labeling Kits are our best option for covalently attaching a fluorophore to small amounts of IgG antibody (~10–20 μg). It is ideal for the efficient labeling of antibodies in serum, ascites fluid, or hybridoma suspensions. Labeled antibodies are ready for use in imaging or flow cytometry applications in as little as 2.5 hours with very little hands on time.

Important Features of Alexa Fluor® APEX® Antibody Labeling Kits:

• Labeled antibodies typically ready to use in 2.5 hours (~15 minutes hands on time)
• Designed to label 10–20 μg of IgG
• Covalent attachment
• Compatible with contaminating proteins or stabilizers like BSA
• No columns needed; everything you need is supplied for 5 separate labelings
• Choose from Alexa Fluor® 488, 555, 568, 594, and 647 dyes, Oregon Green® 488 dye, Pacific Blue™ dye, and Biotin-XX.


Better Results and Workflows With Primary Labeled Antibodies
A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding. Further, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same experiment if they are directly labeled with compatible fluorophores.

Contaminating Proteins or Protein Stabilizers Are Not a Problem
Many IgG antibodies are often available only in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants which can interfere with the amine-reactive labeling reagents. The APEX® Antibody Labeling Kits avoids this by utilizing a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX® antibody labeling tip. Contaminants are simply eluted through the tip, prior to applying the amine-reactive label.

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 Antibody Labeling from A to Z or use our Labeling Chemistry Selection Tool for other choices. To learn more about our various kits read Kits for Labeling Proteins and Nucleic Acids—Section 1.2 in the Molecular Probes® Handbook.

We’ll Make a Custom Antibody Conjugate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom antibody 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.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

Qdot™ 545 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 545 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 545 ITK™ carboxyl quantum dot has emission maxima of ~545 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

EZ-Link™ NHS-PEG Solid-Phase Biotinylation Kit - Mini-Spin Columns (Thermo Scientific™)

Thermo Scientific EZ-Link NHS-PEG Solid Phase Biotinylation Kits provide an on-column approach to amine-targeted antibody biotinylation that provides greater control over the labeling reaction and clean-up process.

Features of the EZ-Link NHS-PEG Solid-Phase Biotinylation Kit:

Suitable for larger-scale reactions—1 to 10 mg antibody
Fast labeling and purification—the entire procedure takes only about one hour
Easy removal of spent and excess labeling reagent—simply wash away the reaction byproducts—no need for dialysis or gel filtration
No dilution effects—solid-phase method allows initially dilute antibodies to be recovered in a smaller volume after labeling
Optimized protocols—specific protocols for antibody ensure appropriate level of labeling (2 to 5 biotins per antibody molecule), minimizing possibility of inactivation caused by overlabeling
High-performance biotin reagent—NHS-PEG4-Biotin uses efficient, amine-specific NHS-ester chemistry and includes hydrophilic polyethylene glycol (PEG) spacer that stabilizes long-term solubility of labeled antibodies
Eight biotin-labeling experiments per kit—Thermo Scientific No-Weigh Packaging ensures that the biotin reagent is fully active for eight separate experiments

This innovative antibody-labeling system uses nickel-chelated agarose to temporarily immobilize antibody molecules via their histidine-rich Fc regions. Once held in place on the resin, the antibody can be biotinylated at primary amines with the high-quality, solubility-stabilizing NHS-PEG4-Biotin reagent. Excess labeling reagent and byproducts are then washed away before recovering the labeled and purified antibody from the resin using a mild imidazole solution. No gel filtration or dialysis is needed.

EZ-Link Solid-Phase Biotinylation Kits use an innovative strategy for antibody biotinylation that simplifies the process in many respects, making successful biotinylation possible for the novice and expert. The strategy involves immobilizing the antibody to a metal-chelated affinity support and performing the biotinylation in the solid phase.

Antibodies bind readily to metal-chelated supports, such as nickel or cobalt, binding primarily through the histidine-rich Fc region of IgG-class antibodies. This feature allows the derivatization chemistry to be performed easily while the antibody is bound to the support. This strategy facilitates reagent delivery and removal of spent reagent. The resulting modified antibody is then selectively removed from the support by elution with a buffered imidazole solution.

The solid-phase approach to antibody biotinylation provides more control over the reaction. For example: Reagent and antibody ratios and reaction times can be adjusted to achieve the proper degree of biotinylation for the intended application.

The kits contain an aqueous soluble amino group-specific reagent (NHS-PEG4-Biotin) that targets accessible amino (-NH2) groups on the surface of the antibody. The format can be applied at the multi-milligram antibody level in the 1 mL column kit (Part No. 21440) or to low microgram antibody levels for biotinylations using the mini-spin column kit (Part No. 21450).

Related Products
EZ-Link™ NHS-PEG Solid-Phase Biotinylation Kit - 1 mL Column

Tetramethylrhodamine (TAMRA) Alkyne (5-Carboxytetramethylrhodamine, Propargylamide), 5-isomer (Invitrogen™)

The red-fluorescent tetramethylrhodamine (TAMRA) alkyne can be reacted with azides via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit (Invitrogen™)

The Click-iT® Plus OPP Alexa Fluor® 647 Protein Synthesis Assay Kit provides a fast, sensitive, and non-radioactive method for the detection of protein synthesis using fluorescence microscopy or high-content imaging. In this assay O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in complete methionine-containing media and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific, and mild click reaction.

Features of the kit include:

• No media change required—works in complete, methionine-containing media, no need to remove cell media
• Multiplex-enabled—Click-iT® Plus technology retains signal from GFP and binding of fluorescent-conjugated phalloidins
• Non-radioactive—an alternative to the traditional 35S-methionine methods
• Works in vivo—published results demonstrate use in vivo for determination of protein translation

The kit contains O-propargyl-puromycin (OPP), which is an alkyne analog of puromycin (also available separately), as well as Alexa Fluor® 647 picolyl azide and all necessary reagents to perform the Click reaction. The OPP is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor® 647 picolyl azide and the Click reaction reagents leads to a chemoselective ligation, or "click" reaction, between the picolyl azide dye and the OPP alkyne, allowing the modified proteins to be detected by imaged-based analysis.

The click reaction uses bioorthogonal (biologically unique) moieties to fluorescently label proliferating cells, helping to produce low backgrounds and high detection sensitivities. Because of the mild reaction conditions, Click-iT® Plus assays detect protein translation events while enabling preservation of cell morphology, the binding of fluorescently-labeled phalloidin, and the fluorescent signal from GFP.

Unlike 35S-methionine, used in traditional methods, OPP is not an amino acid analog, so it can be added directly to cells in complete media or used to determine protein synthesis in vivo.

The kit contains all of the components needed to label and detect the incorporated OPP in newly translated proteins in samples of adherent cells. The kit includes sufficient reagents for the labeling of 25 18 mm × 18 mm coverslips using 1 mL of reaction buffer per test.

ATTO-TAG™ FQ Amine-Derivatization Kit (Invitrogen™)

ATTO-TAG™ FQ (3-2-(furoyl quinoline-2-carboxaldehyde) included in this amine-derivatization kit reacts specifically with primary amines to form conjugates that can be analyzed by electrophoretic or chromatographic methods. The resulting products of FQ are maximally excited at 480 nm or by the 488 nm spectral line of the argon-ion laser and have emission maxima at ~590 nm. The high sensitivity, freedom from background and long-wavelength excitability make these potential reagents for researcher, diagnostic and forensic applications.

EZ-Link™ TFPA-PEG3-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link TFPA-PEG3-Biotin is an efficient, photoactivatable reagent based on tetrafluorophenyl azide for biotinylation and includes a 3-unit polyethylene glycol (PEG) spacer arm.

Features of EZ-Link TFPA-PEG3-Biotin:

Biomolecular labeling—biotinylate proteins, DNA, RNA and many other macromolecules, even if they do not possess primary amines or sulfhydryl groups
Photo-reactive—perfluorophenyl azido group activates upon exposure to ultraviolet light to form covalent bonds with nucleophiles and many other chemical groups
Pegylated—spacer arm contains a hydrophilic, 3-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—best to dissolve in DMSO or DMF before further dilution in aqueous buffers
Long reach —spacer arm (total length added to target) is 33.4 angstroms, minimizing steric hindrance for binding interactions with streptavidin

TFPA-PEG3-Biotin is a photoactivatable reagent for biotinylation of antibodies, proteins and many other kinds of macromolecules. The tetrafluorophenyl azide (TFPA) group activates upon exposure to UV-Light (maximum absorptivity is at 320 nm) to insert covalently at sites containing C-H or N-H bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled molecules stored in solution. The PEG spacer arm also gives this reagent a long and flexible connection to minimize steric hindrance involved with binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency, and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Several different types of photoreactive compounds are available. Aryl azide reagents activate upon exposure to ultraviolet light initiate addition reactions with double bonds, insertion into C–H and N–H sites, or subsequent ring expansion to react with a nucleophile (e.g., primary amines).

Fluorescein-5-Thiosemicarbazide (Invitrogen™)

The amine-containing fluorescein-5-thiosemicarbazide can be reversibly coupled to aldehydes and ketones to form a Schiff base - which can be reduced to a generate stable amine derivative by sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNH3). 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).

EZ-Link™ Maleimide-PEG2-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Maleimide-PEG2-Biotin is a mid-length, maleimide-activated, sulfhydryl-reactive biotinylation reagent that contains a 2-unit ethylene glycol in its spacer arm for increased water-solubility characteristics.

Features of EZ-Link Maleimide-PEG2-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Pegylated—spacer arm contains a hydrophilic, 2-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—can be dissolved directly in aqueous buffers for labeling reactions
Medium length—spacer arm (total length added to target) is 29.1 angstroms

Maleimide-PEG2-Biotin enables simple and efficient biotinylation of antibodies, cysteine-containing peptides and other thiol-containing molecules. The maleimide group reacts specifically and efficiently with reduced thiols (sulfhydryl groups,—SH) at pH 6.5 to 7.5 to form stable thioether bonds. The hydrophilic, 2-unit polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG segment adds length and flexibility to the spacer arm, minimizing steric hindrance involved with binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Maleimide reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

6-JOE, SE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein, Succinimidyl Ester) (Invitrogen™)

This single isomer preparation of the amine-reactive dye 6-JOE, SE has fluorescence characteristics that are red-shifted as compared to fluorescein (excitation/emission is 522/550 nm) and is one of the traditional fluorophores used in automated DNA sequencing.

5-ROX (5-Carboxy-X-Rhodamine, Triethylammonium Salt), single isomer (Invitrogen™)

The carboxylic acid of 5-ROX is used for oligonucleotide labeling and automated DNA sequencing applications. Conjugates of this dye have longer-wavelength spectra than the spectra of Lisaamine™ rhodamine B conjugates, but somewhat shorter-wavelength spectra than those of Texas Red® conjugates.

CMNB-Caged Carboxyfluorescein, SE (5-Carboxyfluorescein-Bis-(5-Carboxymethoxy-2-Nitrobenzyl) Ether, β-Alanine-Carboxamide, Succinimidyl Ester) (Invitrogen™)

Conjugation of the succinimidyl ester of the water-soluble, CMNB-caged carboxyfluorescein, SE to a primary amine on a biomolecule of interest produces an essentially nonfluorescent probe that yields a green-fluorescent fluorescein-labeled product only after ultraviolet illumination and removal of the caging group.

Qdot™ 705 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 705 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 705 ITK™ carboxyl quantum dot has emission maxima of ~705 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

ATTO-TAG™ CBQCA Derivatization Reagent (CBQCA; 3-(4-Carboxybenzoyl)quinoline-2-Carboxaldehyde) (Invitrogen™)

The ATTO-TAG™ CBQCA derivatization reagent (CBQCA; 3-(4carboxybenzoyl)quinoline- 2-carboxaldehyde) reacts specifically with primary amines to form conjugates that can be analyzed by electrophoretic or chromatographic methods. The resulting products of CBQCA are maximally excited at 450 nm or by the 442 nm spectral line of the He-Cd laser and have emission maxima at ~550 nm. In capillary zone electrophoresis (CZE), the sensitivity of detection of ATTO-TAG™ CBQCA conjugates should be in the attomole range (10-18 moles). The high sensitivity, freedom from background and long-wavelength excitability make these potential reagents for researcher, diagnostic and forensic applications.

Pacific Blue™ Succinimidyl Ester (Invitrogen™)

The amine-reactive Pacific Blue™ succinimidyl ester can be used to can be used to create blue-fluorescent bioconjugates with excitation/emission maxima ~410/455 nm that are excitable by the 405 nm spectral line of the blue diode (violet) laser.

View all Pacific Blue™ dye products..

View the Fluorophore Selection Guide.

6-TAMRA (6-Carboxytetramethylrhodamine), single isomer (Invitrogen™)

Tetramethylrhodamine (TMR, TRITC) has been a widely used fluorophore for preparing bioconjugates, especially fluorescent antibody and avidin derivatives used in immunochemistry. Under the name TAMRA, the carboxylic acid of 6-TAMRA has also achieved prominence as a dye for oligonucleotide labeling and automated DNA sequencing applications.

R-Phycoerythrin, Pyridyldisulfide Derivative (Invitrogen™)

The pyridyldisulfide derivative of R-phycoerythrin (R-PE) can be reacted directly with thiolated antibodies, enzymes and other biomolecules to form a disulfide linkage.

Click-iT™ SDP Ester sDIBO Alkyne for Antibody Labeling (Invitrogen™)

The Click-iT SDB Ester 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.

Qdot™ 565 ITK™ Carboxyl Quantum Dots (Invitrogen™)

Qdot® 565 ITK™ carboxyl quantum dots are the ideal starting material for preparing custom conjugates that require high loading of biomolecules. These materials are carboxylate functionalized and can be coupled to amine groups of proteins and modified oligonucleotides using EDC-mediated condensation. The coatings of these probes provides more binding sites than our Qdot® ITK™ amino quantum dots, but lacks PEG linkers that help to prevent non-specific interactions. These materials can be conjugated to X-PEG-amine bi-functional linkers for custom reactivity and higher specificity. Our Qdot® ITK™ carboxyl quantum dots are provided as 8 µM solutions and are available in all 9 Qdot® probe colors.

Important Features of Qdot® ITK™ Carboxyl Quantum Dots:
• Qdot® 565 ITK™ carboxyl quantum dot has emission maxima of ~565 nm
• Extremely photostable and bright fluorescence
• Efficiently excited with single-line excitation sources
• Narrow emission, large Stokes shift
• Available in multiple colors
• Ideal labeling and tracking applications


Properties of Qdot® Nanocrystals
Qdot® probes are ideal for imaging and labeling applications that require bright fluorescent signals and/or real-time tracking. Unique among fluorescent reagents, all nine available colors of Qdot® probes can be simultaneously excited with a single (UV to blue-green) light source. This property makes these reagents excellent for economical and user-friendly multiplexing applications. Qdot® labels are based on semiconductor nanotechnology and are similar in scale to moderately sized proteins.

About the Innovator’s Tool Kit Qdot® ITK™ Reagents
These Qdot® ITK™ probes are ideal for researchers who wish to prepare specific (non-stocked) conjugates for their applications and need customizable conjugation functionality.

Other Forms of Qdot® Nanocrystals are Available
In addition to the carboxyl-derivatized form, we offer Qdot® ITK™ quantum dots with amino and aliphatic hydrocarbon modifications. We’ve also developed a wide range of Qdot® nanocrystals conjugates and labeling kits. Investigate the properties of Qdot® nanocrystals or read the Molecular Probes® Handbook Section 6.6—Qdot® Nanocrystals to find out more.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

DMNP-EDTA (1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N',N'-Tetraacetic Acid), cell impermeant (Invitrogen™)

The caged Ca2+ chelator, DMNP-EDTA (also known as DM-Nitrophen®) upon photolysis, the Kd for Ca2+ increases from 5 nM to 3 mM. Thus, photolysis of DMNP-EDTA complexed with Ca2+ results in a pulse of free Ca2+.

PowerLoad™ Concentrate, 100X (Invitrogen™)

PowerLoad™ is an optimized formulation of nonionic, Pluronic® surfactant polyols for the solubilization of water-insoluble dyes and other materials in physiological media. These surfactants, for instance Pluronic® F-127, have been used to help disperse acetoxymethyl (AM) esters of fluorescent ion indicators such as fluo-4, fura-2, indo-1, fluo-3, and SBFI; they appear to be required for loading of other dyes (e.g. SBFI-AM or PBFI-AM). The use of PowerLoad™ is optional with red shifted calcium indicators and other large molecular weight AM ester dyes, and may also be useful for dispersing other lipophilic probes. The concentration of Pluronic® surfactants in PowerLoad™ is less than 0.2%. PowerLoad™ is effective in combination with water soluble Probenecid (P36400) to aid AM ester dye-loading and retention in cells that actively extrude the de-acetylated form through anion pumps. Together, these reagents allow for maximal loading of dyes with a minimum of effort in both imaging and high throughput screening (HTS) applications. Appropriate controls should be performed to make certain that PowerLoad™ is not altering the membrane properties of the cell.