Kits for Labeling Proteins and Nucleic Acids—Section 1.2

Page Contents

• Kits for Labeling Proteins with a Fluorescent Dye
• Kits for Labeling Proteins with Biotin or Dinitrophenyl (DNP)
• Kits for Labeling Nucleic Acids with a Fluorescent Dye
• Biotin Quantitation Assay Kits
• Ordering Information—All Kits for Labeling Proteins and Nucleic Acids
• Ordering Information—Protein Labeling Kits
• Ordering Information—Nucleic Acid Labeling Kits
• Ordering Information—Zenon Antibody Labeling Kits

We provide a vast number of stand-alone reagents for preparing bioconjugates, most of which are described in detail in other sections of this chapter. This section describes the many specialized kits that we have developed for covalently labeling proteins and nucleic acids with our organic dyes and haptens (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3).

As an alternative to direct conjugation of primary antibodies with our reactive dyes and haptens, we recommend using Zenon technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) to form labeled antibody complexes. Zenon labeling can be completed in minutes with quantitative yield starting with submicrogram quantities of antibody, and the labeling stoichiometry can be easily adjusted to deliver optimum fluorescence output. Although they do not provide covalent labeling, the Zenon Antibody Labeling Kits are listed in Active esters and kits for labeling proteins and nucleic acids—Table 1.2, as well as in Zenon Antibody Labeling Kits—Table 7.7.

APEX Antibody Labeling Kits

APEX Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a convenient method for covalently labeling small amounts (10–20 µg) of IgG antibody with Alexa Fluor dyes, Oregon Green 488 dye or Pacific Blue dye (or with biotin, see below). A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding than secondary antibodies. Furthermore, multiple primary antibodies of the same isotype or derived from the same species can be used in the same immunostaining experiment if they are directly labeled with compatible fluorophores. Many IgG antibodies, however, are often only available in small quantities and may be mixed with stabilizing proteins, such as BSA, that can react nonproductively with the amine-reactive labeling reagents.

APEX Antibody Labeling Kits are specifically designed to allow labeling of small amounts of IgG antibody, even in the presence of contaminants. These kits utilize a solid-phase labeling technique that captures the IgG antibody on resin inside an APEX antibody labeling tip (Figure 1.2.1). Any contaminants, including stabilizing proteins or amine-containing buffers, are eluted through the tip before labeling. After applying the amine-reactive fluorophore to the IgG antibody trapped on the resin, a fluorescent IgG conjugate is formed and subsequently eluted from the resin using elution buffer. The fluorescent IgG conjugate is ready to use in an imaging or flow cytometry assay in as little as 2.5 hours with minimal hands-on time. The typical yield of labeled antibody using this method is between 40 and 80%.

Each APEX Antibody Labeling Kit provides all reagents required to perform five separate labeling reactions of 10–20 µg IgG antibody, including:

• Five vials of amine-reactive fluorescent dye
• Five APEX antibody labeling tips
• Wash, labeling, neutralization and elution buffers
• Dimethylsulfoxide (DMSO)
• Labeling protocol (APEX Antibody Labeling Kits)

For labeling larger amounts of protein, we recommend the Alexa Fluor Microscale Protein Labeling Kits, which are optimized for 20–100 µg samples of proteins between 10,000 and 150,000 daltons; the Alexa Fluor Antibody Labeling Kits, which are optimized for 100 µg samples of mono- or polyclonal antibodies; or the Alexa Fluor Protein Labeling Kits, which are optimized for 1 mg samples of >40,000-dalton proteins.

Figure 1.2.1 Illustration of the use of the APEX antibody labeling tip, provided in APEX Antibody Labeling Kits. A) Applying solutions to the resin in the tip. B) Pushing solutions onto the resin in the tip by attaching the APEX antibody labeling tip to a pipette.

SiteClick Antibody Labeling Kits

The SiteClick system is a modular, click chemistry–mediated method for enzymatically labeling essentially any antibody on its heavy chain N-linked glycans (Figure 1.2.2). This antibody labeling method allows simple and gentle site-selective attachment of detection molecules to heavy chain N-linked glycans—far from the antigen-binding domain—providing excellent reproducibility from labeling to labeling and from antibody to antibody. The SiteClick antibody labeling method prevents disruption of the antigen-binding domain that can occur with traditional amine- or thiol-reactive labeling reagents and eliminates the need to genetically engineer labeling sites into the antibody prior to modification. This site-selective strategy is especially important when labeling monoclonal antibodies that contain lysine residues in or around the antigen-binding domain, as labeling of these sites can disrupt antigen binding. Monoclonal antibodies can also have variable sensitivities to disulfide bond–reducing agents used in reductive cysteine labeling, leaving partially dissociated antibody fragments that result in decreased antibody binding and yield. A number of different detection molecules can be site-selectively attached to the heavy chain glycans—including phycobiliproteins (e.g., R-PE), Qdot probes, fluorescent dyes, metal-chelating compounds, and other small molecules like biotin—allowing multiplex analysis with antibodies from the same species (Figure 1.2.3).

In general, IgG antibodies contain two N-linked glycans attached to specific conserved asparagine residues located in the antibody heavy chain Fc domain. These sugar chains, predominantly complex biantennary glycans with two terminal branches, are structurally quite homogeneous, and the terminal sequences of the glycan branches are highly consistent. Most of the antibody glycan branches terminate with galactose-N-acetylglucosamine (Gal-GlcNAc-) or with N-acetylglucosamine (GlcNAc-). Removal of the terminal Gal residue with β-galactosidase unmasks the majority of terminal GlcNAc labeling sites for the subsequent enzymatic β-galactosyl transferase (GalT) reaction (Figure 1.2.2). Although a small percentage of antibodies also contain terminal sialic acids, our experiments have shown that the additional removal of terminal sialic acid residues produces little difference in the final degree of antibody labeling. After cleavage of terminal Gal residues by β-galactosidase, each N-linked glycan will contain, on average, 2 terminal GlcNAc residues per heavy chain (4 terminal GlcNAc per antibody). In practice, the degree of labeling of mouse and rabbit monoclonal antibodies, as determined by subsequent attachment of fluorescent DIBO dyes, averages between 3 and 4 labels per antibody.

The SiteClick enzymatic labeling approach takes advantage of the modified β-GalT1 enzyme GalT(Y289L), which is substrate permissive and attaches azide-modified N-acetylgalactosamine (GalNAz) selectively to terminal GlcNAc residues on N-linked antibody glycans (Figure 1.2.4). These azide-activated antibodies are then reacted with dibenzocyclooctyne (DIBO)-functionalized molecules in a copper-free click reaction, which produces a strong and stable bond between the DIBO detection molecule and the azide-modified antibody. By coupling GalT(Y289L) enzymatic labeling with a click reaction, the SiteClick system achieves highly efficient and reproducible site-selective labeling of antibodies, regardless of their originating species or antibody class.

Our SiteClick products include R-PE antibody labeling kits, which are designed especially for flow cytometry applications, as well as several Qdot antibody labeling kits that produce conjugates for imaging or flow cytometry applications. The kits are configured to provide an easy-to-follow workflow that will allow novice and experienced scientists alike to obtain efficient antibody labeling every time. And, because antibody glycans are highly conserved, even between different species, the reproducibility of SiteClick labeling for different antibodies is very high, precluding the need to optimize labeling of each newly acquired antibody. Each SiteClick Antibody Labeling Kit provides all reagents required to perform one conjugation reaction of R-PE or Qdot fluorophores to 100–125 µg IgG of purified IgG antibody, including:

• DIBO-modified R-PE or Qdot fluorescent label
• Labeling buffers and additive
• β-Galactosidase
• UDP-GalNAz
• β-1,4-galactosyltransferase (GalT)
• Antibody concentrators and collection tube
• Labeling protocol (SiteClick Antibody Labeling Kits)

The SiteClick antibody labeling kits are compatible with antibodies from a number of different species including, but not limited to, human, rabbit, mouse, rat, goat, hamster, and chicken. Additionally, SiteClick labeling is effective with several antibody classes such as IgG, IgM, and IgY; note that chicken IgY antibodies have 6 heavy chain glycans instead of 2 and therefore can be labeled to a higher extent.

Figure 1.2.2 The SiteClick antibody labeling system. The first step in the SiteClick antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice. The average degree of labeling is 3–3.5 labels per antibody.

Figure 1.2.3 Immunocytochemistry with SiteClick labeled antibodies. HeLa cells were fixed, permeabilized, and incubated with 10 nM Qdot 655 anti–golgin 97 antibody (magenta). This conjugate was generated using the SiteClick Qdot® 655 Antibody Labeling Kit (S10453) and mouse monoclonal anti–golgin-97 antibody (A21270). After antibody incubation, the cells were counterstained with NucBlue Live (R37605) and ActinGreen 488 (R37110) ReadyProbes reagents prior to imaging.

Figure 1.2.4 Site selectivity of SiteClick antibody labeling. A) A β-tubulin monoclonal antibody was azide-activated using the GalT(Y289L) enzyme, labeled with DIBO-functionalized Alexa Fluor 488 dye, and then analyzed by gel electrophoresis (left panel). Once imaged, the same gel was post-stained with SYPRO Ruby Total Protein Gel Stain (S12000, right panel), showing that only the heavy chain of the antibody is click-labeled with the Alexa Fluor 488 DIBO alkyne. B) Anti–β-tubulin mouse monoclonal antibodies were labeled with a small-molecule DIBO-PET chelating agent (L) or left unlabeled (U) (left two lanes) and then post-treated with PNGase F, which selectively cleaves N-linked glycans from asparagine residues (right two lanes). After treatment with PNGase F, both chelator-labeled (L) and unlabeled (U) species are shifted to the same lower molecular weight, confirming the site-selective labeling of heavy chain N-linked glycans.

Alexa Fluor, Pacific Blue and Pacific Orange Antibody Labeling Kits

The Alexa Fluor, Pacific Blue and Pacific Orange Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide researchers with a simple yet efficient means of labeling small amounts of  antibodies with Alexa Fluor dyes or with Pacific Blue or Pacific Orange dye (Figure 1.2.5). These kits contain everything needed to perform five separate labeling reactions containing ~100 µg each of carrier-free antibody samples (although other proteins can be labeled).

Each Antibody Labeling Kit provides:

• Five vials of amine-reactive fluorescent dye (succinimidyl ester or tetrafluorophenyl ester)
• Sodium bicarbonate
• Spin columns and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Antibody Labeling Kits)

Simply dissolve the carrier-free antibody at ~1 mg/mL in the provided buffer, then add it to one of the five vials of amine-reactive dye; no organic solvents are required. Purification of the fluorescent conjugate is accomplished on a size-exclusion spin column optimized for proteins with molecular weight greater than 40,000 daltons. Labeling and purification can be completed in less than 2 hours.

Mouse antibodies in serum, in ascites fluid or diluted with carrier proteins should not be labeled with these kits; however, such antibody preparations can be efficiently labeled with the APEX Antibody Labeling Kits described above or with the Zenon Mouse IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7) described below.


Figure 1.2.5 Illustration of the three simple steps in the protocol for the Alexa Fluor, Pacific Blue and Pacific Orange Antibody Labeling Kits, which provide a convenient method for covalently labeling small amounts of  antibodies.

SAIVI Antibody Labeling Kits

The optimal fluorescent antibody conjugate for in vitro detection assays produces an intense fluorescent signal yet retains the binding affinity and specificity of the unlabeled antibody. When preparing a fluorescent antibody conjugate for in vivo animal imaging, however, the pharmacokinetics of the labeled probe must also be considered. These additional constraints have led to the development of the SAIVI Antibody Labeling Kits for small animal in vivo imaging applications (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3).

SAIVI Antibody Labeling Kits feature reactive far-red and near-infrared Alexa Fluor dyes, along with a labeling protocol specifically designed to produce a suitable degree of labeling (DOL) for in vivo imaging applications. When optimally labeled, a fluorescent antibody conjugate produces an intense, targeted fluorescent signal that persists throughout the in vivo study, without significant redistribution or clearance of the probe.

The conjugation protocol in the SAIVI Rapid Antibody Labeling Kits produces an optimal DOL (~2 fluorophores per antibody) over a sixfold antibody concentration range with no adjustments in reaction volume, dye concentration or antibody concentration necessary. Purification of the dye-labeled antibody is achieved using a simple protocol that can be completed in less than 10 minutes. With these kits, optimally labeled antibodies are ready for applications that require azide-free reagents, such as live-cell imaging or direct injections into animals. SAIVI Rapid Antibody Labeling Kits containing either Alexa Fluor 680 or Alexa Fluor 750 dye (S30045, S30046) provide sufficient reagents for three labeling reactions of 0.5–3 mg of protein each, including:

• Three vials of amine-reactive Alexa Fluor 680 or Alexa Fluor 750 dye
• Sodium bicarbonate
• Regulator solution
• Purification resin and purification columns
• Phosphate-buffered saline (PBS)
• Syringes, syringe filters, column-loading pipettes and catch tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (SAIVI Rapid Antibody Labeling Kits)

To control the DOL of the antibody conjugate, the SAIVI Alexa Fluor 647 Antibody/Protein Labeling Kit (S30044) includes a DOL modulating reagent and instructions for decreasing the DOL from its intrinsic highest value by adding specific amounts of this reagent to the labeling reaction. With this method, antibody preparations with varying ratios of dye to protein can be quickly and reproducibly obtained without significant alteration of labeling or purification conditions, allowing more efficient optimization for in vivo imaging applications. Each kit provides sufficient reagents for three labeling reactions of 1 mg protein each, including:

• Three vials of amine-reactive Alexa Fluor 647 dye
• Sodium bicarbonate
• DOL modulating reagent
• Purification resin and purification columns
• Wash buffer
• Column funnels, column holders, disposable pipettes and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (SAIVI Alexa Fluor 647 Antibody/Protein Labeling Kit)

Alexa Fluor Microscale Protein Labeling Kits

Alexa Fluor Microscale Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a convenient means for labeling small amounts (20–100 µg) of purified protein with the highly fluorescent Alexa Fluor dyes. Spin columns are used to purify the labeled protein, with yields between 60 and 90% depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in as little as 30 minutes.

These kits have been optimized for labeling proteins with molecular weights between 12,000 and 150,000 daltons and contain everything needed to perform three labeling reactions and to separate the resulting conjugates from excess dye. Each Alexa Fluor Microscale Protein Labeling Kit provides:

• Three vials of amine-reactive fluorescent dye (succinimidyl ester or tetrafluorophenyl ester)
• Sodium bicarbonate
• Reaction tubes
• Purification resin and spin filters
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Alexa Fluor Microscale Protein Labeling Kits) 

Easy-to-Use Protein Labeling Kits

Our Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a nearly effortless way to label proteins, especially IgG antibodies, with a fluorescent dye (Figure 1.2.6). Simply add ~1 mg of protein (in a volume of ~500 µL and free of amine-containing buffers such as Tris) to one of the three included vials, which contain a premeasured quantity of amine-reactive dye and a magnetic stir bar. Because the reactive dyes used in these kits are water soluble, no organic solvents are required. Purification is accomplished on a gravity-feed size-exclusion column, which is supplied with the kit. Labeling and purification can be completed in about 2 hours, with very little hands-on time.

Each Protein Labeling Kit provides sufficient reagents for labeling three ~1 mg protein samples of a 150,000-dalton protein (such as an IgG), including:

• Three vials of the amine-reactive fluorescent dye (succinimidyl ester or tetrafluorophenyl ester), each containing a magnetic stir bar
• Sodium bicarbonate
• Gravity-feed columns, a size-exclusion resin and concentrated elution buffer
• Column funnels, foam column holders, disposable pipettes and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Protein Labeling Kits)

Researchers have modified α-synuclein using either the Oregon Green 488 Protein Labeling Kit (O10241) or the Alexa Fluor 594 Protein Labeling Kit (A10239). They intentionally produced fluorescent conjugates with a low degree of labeling (DOL) so as not to perturb oligomerization. When comparing Oregon Green 488 synuclein with Alexa Fluor 594 synuclein, they observed essentially the same results in terms of fibril formation as analyzed by fluorescence polarization. They also detected the Oregon Green 488 synuclein conjugate with mouse monoclonal anti–fluorescein/Oregon Green dye antibody (monoclonal 4-4-20, A6421; Anti-Dye and Anti-Hapten Antibodies—Section 7.4), and gold-labeled anti–mouse IgG antibody using electron microscopy.

Figure 1.2.6 Illustration of the three simple steps in the protocol for the Easy-to-Use Protein Labeling Kits, which provide a convenient method for covalently labeling most proteins.

FluoReporter Protein Labeling Kits

The FluoReporter Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) facilitate research-scale preparation of protein conjugates labeled with some of our brightest fluorescent dyes. Typically, labeling and purifying conjugates with the FluoReporter Protein Labeling Kits can be completed in under 3 hours, with very little hands-on time. First, the amount of dye necessary for the desired protein sample is calculated using the guidelines outlined in the kit protocol. After dissolving the dye in dimethylsulfoxide (DMSO), the calculated amount of dye is added to the protein and the reaction is incubated for 1–1.5 hours. Purification is easily accomplished using convenient spin columns designed for use with proteins of molecular weight greater than 30,000 daltons.

Each FluoReporter Protein Labeling Kits provides sufficient reagents to label 5 to 10 protein samples of 0.2–2 mg each in 200 µL volumes :

• Five vials of the amine-reactive dye
• Dimethylsulfoxide (DMSO)
• Reaction tubes, each containing a stir bar
• Spin columns and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling
  

Zenon Antibody Labeling Kits

The Zenon Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Zenon Antibody Labeling Kits—Table 7.7) are useful for the rapid and quantitative labeling of antibodies with dyes (including phycobiliproteins and their tandem conjugates), biotin or enzymes (Figure 1.2.7). Zenon Antibody Labeling Kits are designed to label intact antibodies in amounts from less than 1 µg to as much as 50 µg, starting with a purified antibody fraction or with a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. The Zenon antibody labeling technology is discussed further in Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3.

Figure 1.2.7 Labeling scheme utilized in the Zenon Antibody Labeling Kits. A) An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment. B) This labeled Fab fragment binds to the Fc portion of the IgG antibody. C) Excess Fab fragment is then neutralized by the addition of a nonspecific IgG, preventing crosslabeling by the Fab fragment in experiments where primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme (such as HRP) or to biotin.

APEX Biotin-XX Antibody Labeling Kit

The APEX Biotin-XX Antibody Labeling Kit (A10495, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a convenient method for covalently labeling small amounts (10–20 µg) of IgG antibody with biotin-XX sulfosuccinimidyl ester (biotin-XX SSE). The water-soluble biotin-XX SSE has a 14-atom spacer that enhances the binding of biotin derivatives to avidin's relatively deep binding sites. APEX Antibody Labeling Kits are specifically designed to allow labeling of small amounts of IgG antibody, even in the presence of contaminants. These kits utilize a solid-phase labeling technique that captures the IgG antibody on resin inside an APEX antibody labeling tip (Figure 1.2.1). Any contaminants, including stabilizing proteins or amine-containing buffers, are eluted through the tip before labeling. After applying the amine-reactive biotin to the IgG antibody trapped on the resin, a biotinylated IgG conjugate is formed and subsequently eluted from the resin using elution buffer. The biotinylated IgG conjugate is ready to use in an imaging or flow cytometry assay in as little as 2.5 hours with minimal hands-on time. The typical yield of labeled antibody using this method is between 40 and 80%.

Each APEX Biotin-XX Antibody Labeling Kit provides all reagents required to perform five separate labeling reactions of 10–20 µg IgG antibody, including:

• Five vials of amine-reactive biotin-XX sulfosuccinimidyl ester (biotin-XX SSE)
• Five APEX antibody labeling tips
• Wash, labeling, neutralization and elution buffers
• Dimethylsulfoxide (DMSO)
• Labeling protocol (APEX Antibody Labeling Kits) 

For labeling larger amounts of protein, we recommend the Biotin-XX Microscale Protein Labeling Kit, which is optimized for 20–100 µg samples of proteins between 10,000 and 150,000 daltons; the FluoReporter Mini-Biotin-XX Protein Labeling Kit, which is optimized for 0.1–3 mg samples of >30,000-dalton proteins; or the FluoReporter Biotin-XX Protein Labeling Kit, which is optimized for 5–20 mg samples.

Biotin-XX Microscale Protein Labeling Kits

The Biotin-XX Microscale Protein Labeling Kit (B30010; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a convenient means for biotinylating small amounts (20–100 µg) of purified protein with biotin-XX sulfosuccinimidyl ester (biotin-XX SSE). Spin columns are used to purify the labeled protein, with yields between 60 and 90% depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in as little as 30 minutes.

This kit, which has been optimized for labeling proteins with molecular weights between 12,000 and 150,000 daltons, contains everything needed to perform three labeling reactions and to separate the resulting conjugates from excess reactive biotin. Each Biotin-XX Microscale Protein Labeling Kit provides:

• Three vials of amine-reactive biotin-XX sulfosuccinimidyl ester (biotin-XX SSE)
• Sodium bicarbonate
• Reaction tubes
• Purification resin and spin filters
• Detailed protocols for conjugation and purification (Biotin-XX Microscale Protein Labeling Kit)

For determining the degree of labeling, the FluoReporter Biotin Quantitation Assay Kit for proteins is available separately (F30751) or in combination with the Biotin-XX Microscale Protein Labeling Kit (B30756). When biotinylating larger amounts of protein, we recommend the FluoReporter Mini-Biotin-XX Protein Labeling Kit, which is optimized for 0.1–3 mg samples of >30,000-dalton proteins, or the FluoReporter Biotin-XX Protein Labeling Kit, which is optimized for 5–20 mg samples.

FluoReporter Mini-Biotin-XX Protein Labeling Kit

The FluoReporter Mini-Biotin-XX Protein Labeling Kit (F6347; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) permits efficient biotinylation of small amounts of antibodies or other proteins with biotin-XX sulfosuccinimidyl ester (biotin-XX SSE). The ready-to-use spin columns provide a convenient method of purifying the biotinylated protein from excess reagents.

Each FluoReporter Mini-Biotin-XX Protein Labeling Kit provides sufficient reagents for five labeling reactions of 0.1–3 mg protein each, including:

• Five vials of amine-reactive biotin-XX sulfosuccinimidyl ester (biotin-XX SSE)
• Reaction tubes, each containing a stir bar
• Purification resin, spin columns and collection tubes
• Dialysis tubing
• Detailed protocols for conjugation and purification (FluoReporter Mini-Biotin-XX Protein Labeling Kit) 
  

DSB-X Biotin Protein Labeling Kit

Our unique DSB-X biotin technology, which is described in detail in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, permits the facile reversal of the biotin–avidin interaction under extremely gentle conditions. DSB-X biotin succinimidyl ester, a derivative of desthiobiotin (Figure 1.2.8) with an additional seven-atom spacer, reacts with amine groups of biomolecules to form stable amides. Like biotin conjugates, the DSB-X biotin conjugate can be detected with any avidin or streptavidin derivative; with DSB-X biotin conjugates, however, this binding is almost totally reversed by addition of free biotin (B1595, B20656; Biotinylation and Haptenylation Reagents—Section 4.2) at neutral pH and normal ionic strength.

The DSB-X Biotin Protein Labeling Kit (D20655; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) contains the reagents required for five protein conjugations of 0.5–3 mg each, including:

• Five vials of amine-reactive DSB-X biotin succinimidyl ester
• Dimethylsulfoxide (DMSO) for dissolving the succinimidyl ester
• Reaction tubes
• Purification resin, spin columns and collection tubes for small-scale purifications
• Dialysis tubing for larger-scale separations
• Detailed protocols for conjugation and purification (DSB-X Biotin Protein Labeling Kit)

Figure 1.2.8 Comparison of the structures of D-biotin (top) and D-desthiobiotin (bottom).

ULYSIS Nucleic Acid Labeling Kits

The ULYSIS Alexa Fluor Nucleic Acid Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3, Molecular Probes nucleic acid labeling kits—Table 8.6) provide a simple, reliable method for producing fluorescent hybridization probes by combining our Alexa Fluor fluorophores with the versatile, patented Universal Linkage System (ULS) platinum-based chemistry developed by KREATECH Biotechnology BV. The ULS technology is based on the use of a platinum dye reagent that forms a stable adduct with the N-7 position of guanine and, to a lesser extent, adenine bases in DNA, RNA, peptide–nucleic acid conjugates (PNA) and oligonucleotides (Figure 1.2.9). In protein contexts, ULS reagents are reactive with cysteine residues and other thiols. The labeling reaction takes only 15 minutes, and separation of the labeled nucleic acids from the unreacted ULS reagent can be accomplished through use of a simple spin-column procedure (Figure 1.2.10).

In addition to ULYSIS Alexa Fluor Nucleic Acid Labeling Kits, we offer the ULYSIS Oregon Green 488 Nucleic Acid Labeling Kit (Labeling Oligonucleotides and Nucleic Acids—Section 8.2).  Each of these ULYSIS Kits provides sufficient reagents for 20 labelings of 1 µg DNA each, including:

• ULS labeling reagent and appropriate solvent
• Labeling buffer
• Deoxyribonuclease I (DNase I), for digesting DNA longer than 1000 base-pairs prior to labeling
• DNase I storage and reaction buffers
• Control DNA from calf thymus
• Nuclease-free H₂O
• Detailed protocols for preparing fluorescent DNA hybridization probes for chromosome in situ hybridization and dot-blot hybridization (ULYSIS Nucleic Acid Labeling Kits)

Probes labeled using the ULYSIS Kits are stable indefinitely and hybridize effectively to target DNA. The ULS method has been used to prepare labeled probes for dot, Southern and northern blot analysis, RNA and DNA in situ hybridization, multicolor FISH, comparative genome hybridization (CGH) and microarray analysis.

Figure 1.2.9 Schematic diagram of the labeling method provided in our ULYSIS Nucleic Acid Labeling Kits. The ULS reagent in the ULYSIS Nucleic Acid Labeling Kits reacts with the N-7 position of guanine residues to provide a stable coordination complex between the nucleic acid and the fluorophore label.

Figure 1.2.10 Nucleic acid labeling method provided in our ULYSIS Nucleic Acid Labeling Kits.

ARES DNA Labeling Kits

The ARES DNA Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3, Molecular Probes nucleic acid labeling kits—Table 8.6) provide a versatile, two-step method for labeling DNA with several of our premier fluorescent dyes (Figure 1.2.11). In the first step, an amine-modified nucleotide, 5-(3-aminoallyl)-dUTP, is incorporated into DNA using conventional enzymatic labeling methods. This step ensures relatively uniform labeling of the probe with primary amine groups. The aminoallyl dUTP substrate used in this reaction is taken up efficiently by reverse transcription or nick translation, for which we provide the protocols; other enzymatic methods are also likely to be compatible. In the second step, the amine-modified DNA is chemically labeled using an amine-reactive fluorescent dye. This chemical reaction varies little in its efficiency from dye to dye, so that it is possible to use any combination of the ARES Kits, with their broad selection of the brightest and most photostable dyes, and obtain consistent DNA labeling. The labeling protocols provided generally result in incorporation of about one dye per 12–15 bases, which we have determined to be optimal for fluorescence in situ hybridization (FISH) and dot-blot hybridization.

Each ARES DNA Labeling Kit provides sufficient reagents for 5 to 10 labelings of 1–5 µg DNA each, including:

• 5-(3-Aminoallyl)-dUTP
• Amine-reactive fluorescent dye and appropriate solvent
• Sodium bicarbonate
• Nuclease-free H₂O
• Detailed protocol for labeling DNA using reverse transcription or nick translation (ARES DNA Labeling Kits)

See Labeling Oligonucleotides and Nucleic Acids—Section 8.2 for a complete description of the ARES Kits, as well as of the FISH Tag DNA and FISH Tag RNA Kits, which employ the same aminoallyl nucleotide labeling method but provide a complete workflow solution for fluorescence in situ hybridization (FISH) applications. Each FISH Tag Kit provides all of the reagents needed for enzymatically incorporating the amine-modified nucleotide (aminoallyl dUTP or aminoallyl UTP) into DNA or RNA, followed by fluorescent labeling with an amine-reactive Alexa Fluor dye and purification of the labeled probe using PureLink nucleic acid purification technology (e.g., PureLink PCR Purification Kit, K3100-01). FISH Tag DNA and FISH Tag RNA Kits are each available as single-color kits containing one of four amine-reactive Alexa Fluor dyes or in a multicolor kit that contains all four reactive Alexa Fluor dyes.

Figure 1.2.11 Schematic diagram of the labeling method provided in our ARES DNA Labeling Kits. The ARES DNA Labeling Kits use a two-step method to label DNA. Step 1) The aminoallyl dUTP is enzymatically incorporated. Step 2) A reactive fluorophore is used to label the incorporated aminoallyl group.

Alexa Fluor Oligonucleotide Amine Labeling Kits

The Alexa Fluor Oligonucleotide Amine Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide the reagents required for labeling synthetic oligonucleotides that have amine groups incorporated at their 5'-terminus. Following purification by standard chromatographic or electrophoretic procedures, these singly labeled oligonucleotides can serve as hybridization or ligation probes for a variety of applications.

Each Alexa Fluor Oligonucleotide Amine Labeling Kit contains sufficient reagents for three labelings of 50 µg each of an amine-modified oligonucleotide, including:

• Three vials of amine-reactive dye
• Dimethylsulfoxide (DMSO)
• Three vials of labeling buffer
• Labeling protocol (Alexa Fluor Oligonucleotide Amine Labeling Kits)

FluoReporter Biotin Quantitation Assay Kit for Biotinylated Proteins

The FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins (F30751) provides a sensitive fluorometric assay for determining the number of biotin labels on a protein. This assay is based on the displacement of a ligand tagged with a quencher dye from the biotin-binding sites of Biotective Green reagent. The FluoReporter biotin quantitation assay can detect from 4 to 80 picomoles of biotin in a sample, providing a 50-fold higher sensitivity than the spectrophotometric HABA biotin-binding assay. Furthermore, unlike the HABA biotin-binding assay, which requires ~1 mg of protein sample, the FluoReporter biotin quantitation assay requires a minimum of 600 ng of a singly biotinylated IgG with molecular weight 150,000 daltons. For proteins of lower molecular weight or multiple biotin labels, less protein can be used. To expose any biotin groups in a multiply labeled protein that are sterically restricted and inaccessible to the Biotective Green reagent, this kit includes protease and an optional protocol for digesting the protein. With this preliminary digestion, biotin assay values agree well with MALDI-TOF determinations. With excitation/emission maxima of 495/519 nm, this assay is compatible with any fluorescence-based microplate reader capable of detecting fluorescein (FITC) or Alexa Fluor 488 dye; it can also be scaled up for fluorometer-based experiments.

Each FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins includes:

• Biotective Green reagent
• Biocytin standard
• Protease
• Concentrated phosphate-buffered saline (PBS)
• Biotinylated goat anti–mouse IgG antibody for use as a positive control
• Detailed protocols (FluoReporter Biotin Quantitation Assay Kit For Biotinylated Proteins)

Sufficient reagents are provided for assaying 5 samples independently using eight wells in triplicate for the standard curve and three dilutions of the sample in triplicate (totaling 33 wells per assay). However, fewer wells may be used to conserve sample and a single standard curve can be used for multiple samples in the same experimental session.