Shop All Protein Labeling Reagents

5(6)-TAMRA (5-(and-6)-Carboxytetramethylrhodamine), mixed isomers Invitrogen™

5(6)-TAMRA contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. This bright, orange-fluorescent dye produces conjugates with absorption/emission maxima of ~555/580 nm.

N-(Biotinoyl)-N'-(Iodoacetyl)Ethylenediamine Invitrogen™

The thiol-reactive biotin iodoacetamide reagent can be used to covalently attach biotin to thiol-containing proteins or thiolated nucleic acids.  The addition of a biotin residue enables the detection with avidin or streptavidin conjugates.  Electrophoretically separated thiol-containing proteins treated with biotin iodoacetamide have been detected in Western blots using an avidin–alkaline phosphatase conjugate.

Alexa Fluor™ 555 NHS Ester (Succinimidyl Ester) 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 NHS ester (or succinimidyl ester) of Alexa Fluor® 555 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® 555 dye
Reactive group: NHS ester
Reactivity: Primary amines on proteins and ligands, amine-modified oligonucleotides
Ex/Em of the conjugate: 555/572 nm
Extinction coefficient: 155,000 cm-1M-1
Spectrally similar dyes: tetramethylrhodamine
Molecular weight: ~1250

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.

LanthaScreen™ Amine Reactive Tb Chelate Thermo Scientific™

As part of the LanthaScreen® TR-FRET toolbox of assay reagents, LanthaScreen® Amine Reactive Tb Chelate labeling reagent is available for assay development. The amine-reactive, isothiocyanate group conjugates to virtually any peptide or protein that contains one or more accessible amine moieties, such as the amine group found on a lysine residue or an unmodified N-terminal amine group of a peptide or protein. Amine-modified DNA oligonucleotides can be labeled as well. Energy transfer from the terbium donor to a suitable acceptor such as fluorescein is readily detected by monitoring an increase in acceptor fluorescence intensity. See the user guide for application-based protocols for labeling peptides containing free amino groups, amine-modified oligonucleotides, and IgG antibodies.

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)

pHrodo™ iFL Red STP Ester (amine-reactive) Invitrogen™

The amine-reactive pH-sensitive pHrodo iFL Green STP Ester dye is an improved version of pHrodo Green dye optimized for the creation of bioconjugates to be used in the study of antibody internalization, endocytosis, and phagocytosis. pHrodo iFL Green dye dramatically increases fluorescence as the pH of its surroundings become more acidic. It is more soluble than the original pHrodo Green dye, making it more useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation.

• Use pH-sensitive pHrodo iFL Green STP Ester 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 red fluorescent markers such as RFP, pHrodo iFL Red STP, and many others

The increase in fluorescence of pHrodo iFL Green dye as the pH changes from basic to acidic correlates with the acidification of intracellular vesicles, making it a valuable tool for the study of endocytosis or phagocytosis and their regulation by environmental factors, drugs, or pathogens. The spectral properties of pHrodo iFL Green dye make it useful for multi-color experiments. pHrodo iFL Green dye can be detected using most standard green-fluorescent filter sets and has been validated for use in a variety of applications, including flow cytometry, fluorescent microscopy, and high content screening (HCS). The lack of fluorescence of pHrodo iFL Green dye in a typical extracellular environment eliminates the need for wash steps or quencher dyes in the experimental workflow.

pHrodo iFL Green STP Ester is an amine-reactive dye that can be used to make pHrodo iFL Green STP bioconjugates in aqueous buffer. The STP ester will react with primary amines on a protein, cell, or virus to create a stable conjugate that can be used in live cell assays or stored for later use.

5-TAMRA (5-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 5-TAMRA has also achieved prominence as a dye for oligonucleotide labeling and automated DNA sequencing applications.

Biocytin (ε-Biotinoyl-L-Lysine) Invitrogen™

Biocytin can be used as microinjectable anterograde and transneuronal tracers that is fixable with aldehyde-based fixatives. Additionally the primary amine on the lysine 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).

Pierce™ Dye Removal Columns Thermo Scientific™

Thermo Scientific Pierce Dye Removal Columns effectively bind to unconjugated fluorescent dye molecules from protein solutions to rapidly purify fluorescent conjugated antibodies and other proteins after labeling reactions.

These Fluorescent Dye Removal Columns enable fast and efficient removal of non-reacted fluorescent dyes from protein labeling reactions. Removing excess dye after a labeling reaction is often difficult and time-consuming but is essential for accurate determination of dye-to-protein ratios. The dye removal resin in this kit is highly specialized to produce exceptional protein recoveries while effectively removing non-conjugated dye. Using the appropriate amount of resin and buffer conditions, almost any fluorescent dye can be removed with this kit.

Features of Dye Removal Columns:

Fast—removes free, unconjugated dye from labeled protein solutions in less than a minute
Specific—purification resin provides outstanding conjugate recovery (75 to 95%)
Customizable—separate resin and microcentrifuge columns allow optimization for different dyes and concentrations of sample
Sample-friendly—processing results in minimal sample dilution

This product has been used successfully to remove excess, unconjugated fluorescein, rhodamine and DyLight Dyes from antibodies following labeling reactions. Samples with dyes in the green spectrum, such as Dylight 549 and rhodamine, might require double processing because they require a high molar excesses for labeling. When using fluorescent dyes other than those described in the product instructions, the ratio of resin to protein/dye mixture will require optimization.

DSB-X™ Biotin 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.

SiteClick™ Antibody Azido Modification Kit Invitrogen™

Create a label-ready site-specific azido-modified antibody without lengthy and inefficient genetic modification using the SiteClick Antibody Azido Modification Kit. SiteClick labeling uses enzymes to specifically attach an azido moiety to the heavy chains of an IgG antibody, ensuring that the antigen binding domains remain unaltered for binding to the antigen target. This site selectivity is achieved by targeting the carbohydrate domains present on essentially all IgG antibodies regardless of isotype and host species. Once azido–modified, a variety of labeled SiteClick sDIBO alkynes are available for attachment to the antibody via Click chemistry at your choice of scale. This provides the flexibility to choose different labels for your antibody depending on your assay.

Features of the SiteClick Antibody Azido Modification Kit:
• Contains everything required to azido-modify any species or isotype of IgG antibody
• Easy-to-follow step-by-step protocol
• Highly efficient, site-specific, reproducible labeling chemistry results in high quality antibody conjugate

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.

EZ-Link™ Maleimide-PEG Solid-Phase Biotinylation Kit - Mini-Spin Columns Thermo Scientific™

The Thermo Scientific EZ-Link Maleimide-PEG Solid-Phase Biotinylation Kit provides an on-column approach to sulfhydryl-targeted antibody biotinylation that provides greater control over the labeling reaction and clean-up process.

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

Ideal for small-scale reactions—0.1 to 1 mg antibody
Fast labeling and purification—the entire procedure takes only about two hours
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 gentle disulfide reduction and appropriate level of labeling to minimize the possibility of inactivation
High-performance biotin reagent—Malemide-PEG2-Biotin uses efficient, sulfhydryl-specific maleimide 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 gently reduced at the hinge-region to produce free sulfhydryls that are then biotinylated with the Maleimide-PEG2-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 sulfhydryl group-specific reagent (Maleimide-PEG2-Biotin) that targets accessible sulfhydryl (-SH) groups on the surface of the antibody. The format can be applied to low microgram antibody levels for biotinylations using the mini-spin column kit.

Related Products
EZ-Link™ Maleimide-PEG Solid-Phase Biotinylation Kit - Prepacked Column

CellTracker™ Deep Red Dye Invitrogen™

CellTracker™ Deep Red is a fluorescent dye well suited for monitoring cell movement or location. After loading the cells, the dye is well retained, allowing for multigenerational tracking of cellular movements. The deep red 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)
• Red excitation/emission spectra (630/650 nm maxima) ideal for multiplexing
• Low cytotoxicity—does not affect viability or proliferation

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

DyLight™ 650 Microscale Antibody Labeling Kit Thermo Scientific™

The Thermo Scientific DyLight 650 Microscale Antibody Labeling Kit is an NHS ester-activated derivative of high-performance DyLight 650 used to fluorescently label antibodies and other proteins that are then used as molecular probes for cellular imaging and other fluorescence detection methods. The microscale kit contains all of the necessary components to perform five separate labeling reactions using 100 µg of IgG—the amine-reactive DyLight 650 NHS-ester in convenient single-use vials as well as purification resin and spin columns for the preparation of ready-to-use conjugate.

DyLight 650 provides vibrant far-red fluorescence with comparable or improved performance over other dyes, including Alexa Fluor™ 647 and Cy5™ dye, for fluorescent applications.The high water solubility of DyLight Fluors means that a high dye-to-protein ratio can be attained without causing precipitation of the conjugates. DyLight 650 Amine-Reactive Dye is also available as a stand-alone reagent.

Features of DyLight 650 NHS Ester:

High performance—DyLight 650 fluoresces brighter than Alexa Fluor 647 and Cy5
Specific—NHS ester-activated dye labels proteins and other molecules at primary amines (-NH2)
Convenient kit sizes—standard and microscale sizes are offered to match your experimental needs
Optimized procedure—following the standard protocol results in antibodies with excellent dye:protein ratios and recovery rates for optimum activity and fluorescence labeling

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

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

Typical labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the high solubility of DyLight Fluors permits protein solutions to be added directly to specific amounts of the labeling reagent. This feature allows DyLight 650 Amine-Reactive Dye to be provided in multiple formats with flexible protocols to achieve efficient degrees of labeling.

Related Products
DyLight™ 650 NHS Ester
DyLight™ 650 Antibody Labeling Kit

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.
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