Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3

Our wide selection of Zenon Kits can be mixed and matched in the same experimental protocol and even in the same cell-labeling solution, providing the freedom to experiment with multiple dye–antibody combinations in flow cytometry and imaging applications. In addition, we offer Zenon Mouse IgG₁ Labeling Kits enhanced with TSA technology (Z25090, Z25091), which combine the advantages of Zenon labeling technology with the sensitivity of tyramide signal amplification (TSA). A special web site location (probes.invitrogen.com/products/zenon) has been established for direct access to information on our Zenon labeling technology and a special e-mail address (probeszenon@invitrogen.com) is available to answer questions on this technology and upcoming product releases.

Molecular Probes' Zenon technology has many outstanding features that will undoubtedly make it a preferred method for immunolabeling and will open several new avenues for research and development. These features, which are discussed in detail later in this section, include:

• Labeling of a primary antibody is very fast. The Zenon complex is ready for use in cell-labeling protocols in less than 10 minutes. In the absence of competing antibodies of the same species, the Zenon complex can also be stored for later use.
• Labeling can be quantitative with respect to the primary antibody. Furthermore, the extent of antibody labeling (and thus the intensity of the fluorophore or activity of a conjugated enzyme) can be adjusted by changing the molar ratio of the dye- or enzyme-labeled Fab fragment to the primary antibody. In flow cytometry, this unique property of the Zenon reagents even permits use of the same fluorescent color to label two or more cell populations in the same sample.
• Our dye- and enzyme-labeled Fab fragments have been affinity purified during their preparation to ensure their high affinity and selectivity for the Fc portion of the corresponding primary antibody. Furthermore, our procedure for chemical labeling of the Fab fragments protects the Fc-binding site, resulting in more active labeling reagents.
• Like covalently labeled primary antibodies, the Zenon complexes formed with the Zenon Mouse IgG, Zenon Rabbit IgG, Zenon Goat IgG and Zenon Human IgG Labeling Kits can be used to label mouse, rabbit, goat and human tissue, respectively, without introducing nonspecific background. However, the Zenon complexes are much easier to prepare and more versatile than covalently labeled primary antibodies, and they show higher activity because only the Fc portion of the primary antibody is labeled, leaving the antigen-binding sites available for cell and tissue labeling.
• Zenon reagents provide reliable and reproducible labeling of even submicrogram quantities of a primary antibody, resulting in very little waste of valuable reagents. Furthermore, the Zenon Labeling Kits permit easy and efficient labeling of antibodies that are not commercially available as direct conjugates.
• Because it is based on immunoselectivity, the Zenon labeling method does not require removal of exogenous proteins such as serum albumin or amine-containing buffers from the antibody prior to complex formation, as would be necessary with any chemical labeling procedure.
• Zenon 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 Zenon Labeling Kits (Molecular Probes' Zenon Labeling Kits - Table 7.14). Consequently, there is no need to purchase several directly conjugated primary antibodies for multicolor experiments.
• We have determined that the fluorescence intensity, enzymatic activity and utility of the Fab–antibody complexes formed with the Zenon Labeling Kits are usually similar to the properties of directly conjugated primary antibodies. The fluorescence intensity will depend to some extent on the molar ratio of Fab fragment to antibody used during labeling.
• It should be possible to automate the preparation of an optimal Zenon complex, which may make it practical to label minute amounts of antibodies directly in microplates or in miniaturized devices.
• Essentially any dye or other detectable label can be used in the preparation of a Zenon labeling reagent. The Zenon Antibody Labeling Kits containing our premier Alexa Fluor dyes, phycobiliproteins, horseradish peroxidase (HRP), alkaline phosphatase, biotin and other labels are available (Molecular Probes' Zenon Labeling Kits - Table 7.14).

Figure 7.56 Labeling scheme utilized in the Zenon Antibody Labeling Kits. An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment (panel A). This labeled Fab fragment binds to the Fc portion of the IgG antibody (panel B). Excess Fab fragment is then neutralized by the addition of a nonspecific IgG (panel C), 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.

Zenon Immunolabeling Is Rapid and Quantitative

Labeling of a suitable primary antibody with the Zenon Antibody Labeling Kits is very fast. The Zenon labeling reagent contains dye- or enzyme-labeled Fab fragments that have been affinity purified to ensure their high affinity for the Fc portion of the corresponding primary antibody. We recommend incubation of the Zenon labeling reagent with the appropriate primary antibody for five minutes, followed by a five-minute blocking reaction of any remaining labeled Fab fragments with excess nonspecific IgG. However, the actual rates of the complex formation and IgG blocking steps are likely to be even faster and may occur within the mixing time of the components (Figure 7.57).

Figure 7.57 Formation of antibody–Fab complexes. An anti-biotin mouse IgG₁ monoclonal antibody (A11242) was mixed with the Zenon Alexa Fluor 488 labeling reagent (a component of Kit Z25002) for varying time intervals before the reaction was quenched by the addition of excess mouse IgG blocking reagent. The quenched reactions were then added to a microplate well containing biotinylated bovine serum albumin and incubated for 20 minutes. After washing, the fluorescence of the remaining bound signal was measured. Binding was found to be essentially complete in less than five minutes. Three trials are shown, along with a control () where no labeling reagent was added.

Fab–antibody complex formation can be quantitative with respect to the primary antibody (Figure 7.58). Furthermore, the degree of labeling (and thus the intensity of the fluorophore or activity of a conjugated enzyme) can be adjusted to some extent by changing the molar ratio of the labeled Fab fragment to the primary antibody. We find that approximately equal weights of a dye-labeled Fab fragment generated from a goat anti-Fc antibody (MW ~50,000 daltons) and the intact mouse primary antibody (MW ~150,000 daltons) — a 3:1 molar ratio of the labeled Fab fragment to the primary antibody — yields an Fab–antibody complex that is suitable for most applications. Thus, the 50 µg of labeled Fab fragments in the Zenon labeling reagent provided in the organic dye–based Zenon Labeling Kits is sufficient for labeling approximately 50 µg of the corresponding intact IgG antibody; we have defined "one labeling" in all of our Zenon Labeling Kits as the amount of Zenon labeling reagent required for labeling 1 µg of an intact primary antibody. Increasing the molar (or weight) ratio of the Zenon labeling reagent to the antibody can yield a somewhat brighter conjugate and ensure quantitative utilization of the primary antibody, whereas decreasing the ratio can yield a somewhat less fluorescent complex.

Figure 7.58 High-performance size-exclusion chromatographic analysis of the Zenon Alexa Fluor 488 labeling reagent (a component of Kit Z25002) binding to a mouse IgG₁ antibody. The Zenon labeling reagent peak appears at 38 minutes; the mouse IgG₁ peak appears at 33 minutes. When combined at a molar ratio of ~5:1 (Zenon labeling reagent:IgG₁), the IgG₁ antibody is quantitatively converted to a labeled complex, which appears as a peak at 29 minutes.

The ability to adjust the fluorescence intensity (or enzymatic activity) of a labeled complex is a feature of the Zenon technology that is not at all practical with direct chemical labeling of antibodies. This property also permits the researcher to rapidly optimize the best complex for their experiment, rather than depending on the quite variable degree of substitution that is typical of covalently labeled antibodies from different commercial sources or from chemical labeling in the research laboratory. A molar ratio of three moles of the Zenon labeling reagent to one mole of the primary antibody is also suitable for labeling with the phycobiliprotein- and enzyme-derived Zenon labeling reagents. The Zenon Labeling Kits containing a phycobiliprotein- or enzyme-labeled Fab fragment include sufficient reagents for labeling ~25 µg of an intact primary antibody; the Zenon Labeling Kits containing an Alexa Fluor dye–phycobiliprotein tandem conjugate include sufficient reagents for labeling ~10 µg of a primary antibody.

Zenon Technology Simplifies the Use of Multiple Antibodies of the Same Isotype in the Same Protocol

Unlike detection with secondary antibodies, the Zenon immunolabeling technology allows staining of a cell or tissue sample with multiple antibodies of the same isotype. Our affinity purification of the Zenon labeling reagent (which we perform subsequent to dye or enzyme conjugation) ensures that the formation of the Fab–antibody complex is stable. Reversal of Zenon complex formation by the excess of nonspecific IgG that is used to quench any uncomplexed Zenon labeling reagent does occur (Figure 7.59), particularly at temperatures above room temperature. However, the stability of the 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. Zenon complexes can be combined with each other or with directly labeled primary antibodies, with labeled secondary antibodies to primary antibodies derived from other species, with antibodies that lack an Fc fragment and with avidin–biotin techniques (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) for multiplexed immunolabeling. Some crossreactivity of the Zenon labeling reagent with the Fc portion of antibodies from other species may occur unless the excess Zenon labeling reagent is captured with the soluble nonspecific IgG included in the Zenon Labeling Kits. Alternatively, the excess Zenon labeling reagent from the Zenon Mouse IgG Labeling Kits can be captured and removed entirely from the labeled complexes by agarose-immobilized mouse IgG (M25500).

Zenon Immunolabeling Is Reliable, Even with Very Small Quantities of Reagents

Formation of the Fab–antibody complex with the Zenon Labeling Kits is extremely reliable and reproducible, even with very small (submicrogram) amounts of primary antibody. Successful chemical labeling of submicrogram quantities of an antibody with a succinimidyl ester or an isothiocyanate of a dye is just not possible; chemical labeling and purification of proteins usually requires at least 100 µg of the carrier-free protein. Furthermore, because submicrogram amounts of an immunolabeling complex may be all that are required for an experiment, there is absolutely no waste of expensive or difficult-to-obtain antibodies when using the Zenon Labeling Kits. Additionally, optimization of the degree of labeling by a dye or an enzyme is trivial, as compared with any chemical labeling method. Although Molecular Probes defines "one labeling" in its Zenon Labeling Kits as the amount of Zenon labeling reagent required for labeling 1 µg of the primary antibody, we routinely label about 0.4 µg of the primary antibody dissolved in 2 µL of a buffer.

Unfortunately, many providers of mouse monoclonal antibodies do not indicate how much of the pure antibody they provide in their products. Furthermore, these antibodies are frequently supplied diluted with an albumin or other carrier protein, which precludes estimation of the antibody concentration from the optical density at 280 nm. However, the ease of preparing Zenon complexes makes it practical to rapidly optimize the labeling reagent, even when the amount of primary antibody in the sample is unknown.

Zenon technology should be readily adaptable for use in microplates or miniaturized devices. Microfluidic delivery technology should permit automated formation of Zenon complexes and staining of cells and tissues using less than nanograms of a Zenon labeling reagent and the antibody for high-throughput applications. Because the Zenon labeling reagent is monovalent, the Zenon complexes are not immunoprecipitated during the labeling.

Zenon Immunolabeling Does Not Require Antibody Pre-Purification

Unlike chemically modifying antibodies, labeling antibodies with the Zenon Labeling Kits does not require removal of exogenous proteins such as serum albumin from the antibody. Serum albumin is frequently added to laboratory-derived or commercially supplied antibodies to help preserve the activity of dilute solutions of antibodies. Because the Zenon labeling reagent selectively binds only to the Fc portion of the primary antibody, there should be limited or no effect of exogenous proteins that do not have an Fc fragment. Furthermore, the Zenon labeling reagents function equally well when used to label a purified antibody or a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. Furthermore, Zenon labeling is usually quite successful with even dilute solutions of a primary antibody, as well as with antibodies that are dissolved in amine-containing buffers such as Tris.

Zenon Technology Is Versatile and Cost-Effective

Once conjugated to the appropriate Fab fragment, essentially any dye or other detectable label can be used in the form of a Zenon labeling reagent. We currently offer Zenon Labeling Kits containing a wide selection of different fluorescent labels (Molecular Probes' Zenon Labeling Kits - Table 7.14) whose spectra span the ultraviolet, visible and near-infrared (full spectra of most of these dyes are in A Wide Variety of Protein Conjugates - Section 7.1). The Zenon technology makes it particularly easy to change fluorescent colors or detection methodologies by simply using a different Zenon labeling reagent from our extensive selection of Zenon Labeling Kits (Molecular Probes' Zenon Labeling Kits - Table 7.14). There is no more need to purchase multiple direct conjugates of the same mouse primary antibody (such as with the cluster of differentiation (CD) antibodies) just to perform multicolor flow cytometry (Figure 7.62) and imaging protocols. Furthermore, Zenon labeling utilizes an unlabeled primary antibody, which is usually less expensive than the directly conjugated antibody, particularly for direct conjugates of primary antibodies with phycobiliproteins and enzymes.

Figure 7.62 Human peripheral blood lymphocytes were stained with the following three antibodies: an anti-CD3 mouse IgG₁ antibody (A21330) prelabeled with the Zenon Alexa Fluor 647 Mouse IgG₁ Labeling Kit (Z25008), an anti-CD4 mouse IgG₁ antibody (A21334) prelabeled with the Zenon R-Phycoerythrin Mouse IgG₁ Labeling Kit (Z25055) and an anti-CD8 mouse IgG_2a antibody (A21338) prelabeled with the Zenon Alexa Fluor 488 Mouse IgG_2a Labeling Kit (Z25102). Panels A and B show that cells can be separated by plotting the orange-fluorescent versus green-fluorescent signal or red-fluorescent versus orange-fluorescent signal, respectively, demonstrating that the Zenon label does not transfer to other antibodies in the same sample. The samples were analyzed on a Coulter Elite flow cytometer using 488 nm excitation for R-phycoerythrin and the Alexa Fluor 488 dye, and 633 nm excitation for the Alexa Fluor 647 dye.

The Zenon Labeling Kits that include R-phycoerythrin and allophycocyanin conjugates of the appropriate Fab fragment permit the efficient and cost-effective indirect labeling of a wide variety of primary antibodies for flow cytometry applications, including many of the CD antibodies that currently are not commercially available as direct conjugates. Molecular Probes has also developed Zenon Labeling Kits that utilize our Alexa Fluor dye–phycobiliprotein tandem conjugates (Phycobiliproteins - Section 6.4; Figure 6.34, Figure 6.37) to further expand the range of labels available (Molecular Probes' Zenon Labeling Kits - Table 7.14).

Zenon Mouse IgG, Zenon Rabbit IgG and Zenon Human IgG Labeling Kits are also available for labeling antibodies with HRP or alkaline phosphatase (Molecular Probes' Zenon Labeling Kits - Table 7.14). Several applications of these Zenon Labeling Kits for cytochemistry and histochemistry are described below. Zenon Biotin-XX and DSB-X Biotin Antibody Labeling Kits are also available (Molecular Probes' Zenon Labeling Kits - Table 7.14). Our exclusive 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 completely reversible binding of avidin and streptavidin conjugates at neutral pH and in physiologically compatible buffers. Custom Zenon labeling reagents and Zenon Labeling Kits are also available from Molecular Probes; please contact our Custom and Bulk Sales Department.

The fluorescence intensity or enzymatic activity of a Zenon complex prepared using our protocol (Zenon(R) Mouse IgG Labeling Kits, Zenon(R) Rabbit IgG Labeling Kits) is usually similar to that of the corresponding directly labeled conjugate of the primary antibody, although it is typically somewhat lower than the intensity that can be obtained using a labeled secondary antibody. If necessary, the sensitivity of the assay can be greatly increased through use of a Zenon complex in combination with a signal amplification method such as our TSA or ELF technology (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3); see below for a description of our Zenon Mouse IgG₁ Labeling Kits enhanced with TSA technology (Z25090, Z25091).

Zenon Antibody Labeling Kits for Mouse IgG, Rabbit IgG, Goat IgG and Human IgG

Molecular Probes currently offers Zenon Labeling Kits for six classes of primary antibodies: Zenon Mouse IgG₁ Labeling Kits, Zenon Mouse IgG_2a Labeling Kits, Zenon Mouse IgG_2b Labeling Kits, Zenon Rabbit IgG Labeling Kits, Zenon Goat IgG Labeling Kits and Zenon Human IgG Labeling Kits. These Zenon Labeling Kits, all of which are listed in Molecular Probes' Zenon Labeling Kits - Table 7.14, are of three types:

• Zenon Labeling Kits that contain an affinity-purified 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 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3), to our Patented Marina Blue, Pacific Orange, Pacific Blue, Oregon Green 488 and Texas Red-X dyes or to fluorescein, biotin-XX or DSB-X biotin (desthiobiotin).
• Zenon Labeling Kits with a phycobiliprotein-labeled Fab fragment of a goat anti-Fc antibody (Phycobiliproteins - Section 6.4) — including Zenon Labeling Kits containing one of our five tandem conjugates of R-PE or APC with the long-wavelength Alexa Fluor dyes — for facile labeling of primary antibodies, particularly for applications in multicolor flow cytometry.
• Zenon Labeling Kits containing HRP or alkaline phosphatase conjugates for use in any enzyme-amplified protocol, including immunohistochemical applications, TSA (Tyramide Signal Amplification (TSA) Technology - Section 6.2), ELF (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3) and ELISAs (Secondary Immunoreagents - Section 7.2); see below for a description of our Zenon Mouse IgG₁ Labeling Kits enhanced with TSA technology (Z25090, Z25091).

In addition to the above Zenon Labeling Kits, we have available three different types of Zenon Tricolor Labeling Kits for mouse IgG antibodies, rabbit IgG and human IgG antibodies (Molecular Probes' Zenon Labeling Kits - Table 7.14). The Zenon Tricolor Labeling Kit #1 contains 10 µg each of the Alexa Fluor 488, Alexa Fluor 555 and Alexa Fluor 647 Zenon labeling reagents. This kit is designed for optimal triple-antibody staining with confocal laser-scanning microscopes equipped with an Ar–Kr mixed-gas laser, but it can also be used with any suitably equipped fluorescence microscope. The Zenon Tricolor Labeling Kit #2 contains 10 µg each of the Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 594 Zenon labeling reagents, yielding simultaneous blue-, green- and red-fluorescent immunostaining that is useful with almost any fluorescence microscope. The Zenon Tricolor Labeling Kit #3 is especially suitable for flow cytometry applications and contains 10 µg each of the Alexa Fluor 488, R-phycoerythrin (R-PE) and Alexa Fluor 647–R-PE Zenon labeling reagents, which are all excited efficiently with an argon-ion laser and have minimal spectral overlap (Figure 6.34).

The subtype-selective Zenon Mouse IgG₁, IgG_2a and IgG_2b Labeling Kits can presumably be used with any mouse IgG₁, IgG_2a and IgG_2b antibody, respectively, including those available from Molecular Probes (Molecular Probes' mouse IgG{1} monoclonal antibodies - Table 7.15, Molecular Probes' mouse IgG{2a} monoclonal antibodies - Table 7.16, Molecular Probes' mouse IgG{2b} monoclonal antibodies - Table 7.17). Similarly, the Zenon Rabbit IgG Labeling Kits, Zenon Goat IgG Labeling Kits and Zenon Human IgG Labeling Kits are designed for use with the rabbit IgG antibodies (Molecular Probes' rabbit antibodies - Table 7.18), goat IgG antibodies and human IgG antibodies from Molecular Probes, as well as from any other commercial or research laboratory source.

Zenon Antibody Labeling Kits Enhanced with TSA Technology

The Zenon Mouse IgG₁ Labeling Kits enhanced with TSA technology provide exceptional target-identification capabilities. These kits provide the necessary reagents from both the Zenon Horseradish Peroxidase Mouse IgG₁ Labeling Kit and the corresponding Alexa Fluor TSA Kits for researchers who want both the ease of labeling mouse IgG₁ antibodies with Zenon labeling reagents and the signal amplification afforded by the use of the TSA technology. We currently offer enhanced Zenon Kits containing either the green-fluorescent Alexa Fluor 488 tyramide or the red-orange–fluorescent Alexa Fluor 568 tyramide (Z25090, Z25091). Each kit provides sufficient reagents for 25 labelings, including:

• Zenon HRP mouse IgG₁ labeling reagent
• Zenon mouse IgG blocking reagent
• Alexa Fluor 488 tyramide (in Kit Z25090) or Alexa Fluor 568 tyramide (in Kit Z25091)
• DMSO
• TSA blocking reagent
• TSA amplification buffer
• Hydrogen peroxide (H₂O₂)
• A detailed protocol for Zenon complex formation and fluorescent tyramide labeling (Enhanced Zenon(R) Mouse IgG Labeling Kits)

The Zenon HRP mouse IgG₁ labeling reagent contains Fab fragments of goat IgG antibodies directed against the Fc portion of intact mouse IgG₁ antibodies. These Fab fragments have been purified to ensure their selectivity for the Fc portion of the mouse IgG₁ antibody and then labeled with HRP. This Zenon HRP mouse IgG₁ labeling reagent is simply mixed with any mouse IgG₁ primary antibody to form the Fab–mouse IgG₁ complexes, which can be used for immunolabeling similar to that of primary antibodies covalently labeled with HRP. TSA technology — an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein (Tyramide Signal Amplification (TSA) Technology - Section 6.2) — is then used to detect the target-bound Fab–mouse IgG₁ complex. Each HRP label on the Fab–mouse IgG₁ complexes can activate multiple copies of the Alexa Fluor tyramide to produce short-lived tyramide radicals that are highly reactive with nucleophilic residues near the interaction site, yielding an amplified fluorescent signal with minimal diffusion.

Use and Applications of Zenon Technology

With the exception of our Zenon Tricolor Labeling Kits, our Zenon Labeling Kits contain sufficient reagents for 50 labelings (with the low molecular weight dye–derived Zenon labeling reagents), 25 labelings (with the R-PE–, APC-, HRP- and alkaline phosphatase–derived reagents) or 10 labelings (with the Zenon labeling reagents containing one of five different Alexa Fluor dye–phycobiliprotein tandem conjugates). The Zenon Tricolor Labeling Kits #1, #2 and #3 contain sufficient reagents for 10 labelings of the same or different antibodies with each of three different Alexa Fluor dyes. "One labeling" is defined as the amount of Zenon labeling reagent required for labeling 1 µg of an intact mouse, rabbit, goat or human IgG antibody. However, even smaller (or larger) quantities of a primary antibody can be reliably labeled with any of the Zenon labeling reagents. In the case of the Zenon Labeling Kits containing low molecular weight dyes (Molecular Probes' Zenon Labeling Kits - Table 7.14), approximately equal weights of the dye-conjugated Fab fragment and the primary antibody are used, which gives an approximately 3:1 molar ratio of the Zenon labeling reagent to the primary antibody. Our HPLC analysis of the complex indicates that our procedure (Zenon(R) Mouse IgG Labeling Kits, Zenon(R) Rabbit IgG Labeling Kits) yields almost complete labeling of the primary antibody in the solution, apparently within the mixing time (Figure 7.57). Use of a 5:1 molar ratio of the Zenon labeling reagent gives an apparent 100% conversion of the primary antibody to the labeling complex (Figure 7.58) and a somewhat brighter total fluorescence when compared with the fluorescence of Zenon complexes prepared using a 3:1 ratio. The total fluorescence intensity of the Zenon labeling complex can be raised or lowered to some degree by using a higher or lower ratio of the Zenon labeling reagent to the primary antibody.

For single-color labeling, it is usually not necessary to block any residual Zenon labeling reagent that has not complexed with the primary antibody; however, in applications that involve multiple antibodies of any type (including antibodies from other species or of other isotypes that may react to a small degree with the Zenon labeling reagent), adsorption of residual Zenon labeling reagent is essential to avoid crossreactivity. Adsorption can be done with a solution of soluble nonspecific IgG, which is included in all of the Zenon Labeling Kits. Alternatively, the excess Zenon labeling reagent from the Zenon Mouse IgG Labeling Kits can be quickly adsorbed and removed entirely from the labeled complexes using agarose-immobilized mouse IgG (M25500), which is available separately. Our mouse IgG agarose is supplied with tested protocols (Mouse IgG Agarose) for its use in combination with our Zenon technology. When the Zenon complexes are in a solution containing the Zenon labeling reagents adsorbed onto soluble nonspecific IgG, they should be used for staining within an hour to avoid possible transfer of the Zenon labeling reagent to the excess nonspecific IgG. Zenon complexes can be used with standard immunolabeling techniques; staining with Zenon complexes can be performed sequentially or combined with each other or with additional dye- or enzyme-labeled primary antibody conjugates in a one-step, multiparameter labeling protocol. Fixation with aldehyde-based fixatives following staining is recommended to prevent transfer of the Zenon label between antibodies.

We have demonstrated the utility of Zenon staining for imaging in an assortment of cells (, , , , , , , , ) and some tissues (). As with the use of any antibody conjugate in tissues, staining with Zenon complexes requires good accessibility of the antibody to the target, which can be affected by the specific conditions of fixation and permeabilization. Zenon complexes of intact primary antibodies have somewhat higher molecular weights than direct conjugates of antibodies, potentially reducing the accessibility of the complexes to tissue antigens in some cases.

The Zenon Allophycocyanin and R-Phycoerythrin Antibody Labeling Kits and the Zenon Horseradish Peroxidase and Alkaline Phosphatase Antibody Labeling Kits provide a means to label a primary mouse IgG, rabbit IgG or human IgG antibody with these important labels. The Zenon labeling method is, by far, much faster and easier than the chemical methods that we recommend for crosslinking unlike proteins (Chemical Crosslinking Reagents - Section 5.2; Figure 5.4, Figure 5.6) and results in a much higher (essentially quantitative with respect to the primary antibody) yield of the labeled antibody complex. The Zenon Horseradish Peroxidase and Alkaline Phosphatase Antibody Labeling Kits provide an extremely easy means to amplify the signal when combined with our many TSA reagents (Tyramide Signal Amplification (TSA) Technology - Section 6.2, Tyramide Signal Amplification (TSA) Kits - Table 6.1) or our Enzyme-Labeled Fluorescence (ELF 97) Kits (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3), respectively. The Zenon labeling reagents that contain R-PE, APC, Alexa Fluor dye–phycobiliprotein tandem conjugates, HRP or alkaline phosphatase must not be frozen.

Figure 5.4 SPDP derivatization reactions. SPDP (S1531) reacts with an amine-containing biomolecule at pH 7 to 9, yielding a pyridyldithiopropionyl mixed disulfide. The mixed disulfide can then be reacted with a reducing agent such as DTT (D1532) or TCEP (T2556) to yield a 3-mercaptopropionyl conjugate or with a thiol-containing biomolecule to form a disulfide-linked tandem conjugate. Either reaction can be quantitated by measuring the amount of 2-pyridinethione chromophore released during the reaction.

Figure 5.6 Two-step reaction sequence for crosslinking biomolecules using the heterobifunctional crosslinker SMCC (S1534).

Important Information on Zenon Technology

As our Zenon antibody labeling technology is applied to different experimental systems and protocols, we are compiling a bibliography (Bibliography for Z25999) containing journal articles that cite the use of the Zenon Kits; continuously updated copies are available upon request from our Technical Assistance Department or through this web site. These articles can serve as guides for developing specific applications and protocols. We have extensively tested the Zenon technology with various primary antibodies, particularly in flow cytometry (Figure 7.62) and imaging (, , , , , , , ) applications and found it to be generally reliable, with no more (and sometimes less) background staining than typically occurs with directly conjugated primary antibodies. We have so far done only limited evaluation of Zenon labeling in tissues but will report on these results as they become available.

Because of the importance and uniqueness of our Zenon technology, we maintain a direct link to the latest information on these Zenon products and their applications at our web site. This site includes links to complete Zenon Labeling Kit product lists and information on applications, protocols, pictures and publications of the Zenon technology. In addition, we offer direct e-mail access to request answers to questions on Zenon technology or new product development, which can be obtained by contacting probeszenon@invitrogen.com. We particularly welcome feedback on successes and any problems encountered in using the Zenon technology.

Our Zenon technology is the subject of several Patents and patent applications. Licenses are available from Molecular Probes for commercial resale or high-volume uses of the Zenon labeling reagents and methods that use these reagents. Bulk discounts are available for multiple-unit purchases of the Zenon Labeling Kits. The stand-alone Zenon labeling reagents are not available in bulk, except to licensees of our Zenon technology.