Secondary Immunoreagents—Section 7.2

Page Contents

• Species-Specific Secondary Antibodies
• Image-iT FX Signal Enhancer
• Alexa Fluor Signal Amplification Kits
• Fluorescence- and Chemiluminescence-Based ELISA Kits
• Fluorescence-Based Western Blot Kits
• Gold-Labeled Immunoreagents
• Protein A and Protein G Conjugates
• Ordering Information

We provide an extensive selection of Molecular Probes secondary immunoreagents for use in fluorescence microscopy, flow cytometry and microplate assays, as well as protein and nucleic acid blots and microarrays. Because of their brightness and photostability, the Alexa Fluor conjugates are superior to most conventional fluorescent secondary reagents (Figure 7.2.1, Figure 7.2.2, Figure 7.2.3) and are the detection reagents of choice for many immunofluorescence microscopy, in situ hybridization and bead-based applications. Properties of the organic fluorophores we use to prepare our conjugates are described in detail in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1. We also offer Qdot secondary antibody conjugates (Qdot nanocrystal secondary antibody conjugates—Table 6.11), which combine the fluorescence characteristics of Qdot nanocrystals with the selective binding of F(ab’)₂ fragments from affinity-purified secondary antibodies; the physical and spectroscopic properties of Qdot nanocrystals are discussed in Qdot Nanocrystals—Section 6.6. In addition to our extensive line of species-specific antibodies, isotype-specific antibodies and F(ab')₂ fragments (Antibody Structure and Classification—Note 7.1), we prepare fluorescent conjugates of protein A and protein G—bacterial proteins that bind with high affinity to the Fc portion of various classes and subclasses of immunoglobulins from many species.

The next section of this chapter is devoted to our Zenon antibody labeling technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3). The Zenon antibody labeling technology uses affinity-purified dye- or enzyme-labeled Fab fragments of Fc-specific anti-IgG antibodies for the rapid and quantitative labeling of an intact mouse, rabbit, goat or human IgG antibody. The Zenon labeling method has several advantages over use of the secondary antibodies discussed in this section, including:

• The Zenon labeling method is suitable for quantitatively labeling submicrogram amounts of an antibody in a few minutes and is not affected by the presence of non-antibody proteins or amine-containing buffers in the sample.
• Multiple antibodies derived from the same species can be used in the same experiment.
• The conjugate's degree of substitution and brightness can be easily adjusted.
• The Zenon labeled antibody complexes can be combined with labeled primary and secondary antibodies in most protocols.

The fluorescence intensity of the Zenon labeled antibody complexes may be two- to threefold less than that of labeled secondary antibodies.

Figure 7.2.1 Brightness comparison of Molecular Probes Alexa Fluor 488 goat anti–mouse IgG antibody with Cy2 goat anti–mouse IgG antibody from Jackson ImmunoResearch. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either Alexa Fluor 488 or Cy2 goat anti–mouse IgG antibody at equal concentrations. Red blood cells were lysed, and the samples were analyzed with a flow cytometer equipped with a 488 nm argon-ion laser and a 525 ± 10 nm bandpass emission filter.

Figure 7.2.2 Brightness comparison of Molecular Probes Alexa Fluor 555 goat anti–mouse IgG antibody with Cy3 goat anti–mouse IgG antibody conjugates commercially available from several other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either the Alexa Fluor 555 or Cy3 goat anti–mouse IgG antibody at equal concentrations. Red blood cells were lysed and the samples were analyzed with a flow cytometer equipped with a 488 nm argon-ion laser and a 585 ± 21 nm bandpass emission filter.

Figure 7.2.3 Brightness comparison of Molecular Probes Alexa Fluor 647 goat anti–mouse IgG antibody with Cy5 goat anti–mouse IgG antibody conjugates commercially available from other companies. Human blood was blocked with normal goat serum and incubated with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and incubated with either Alexa Fluor 647 or Cy5 goat anti–mouse IgG antibody at equal concentrations. Red blood cells were lysed and the samples were analyzed with a flow cytometer equipped with a 633 nm He-Ne laser and a longpass emission filter (>650 nm).

Fluorescent Anti-IgG Antibodies

We offer fluorescent secondary antibody conjugates directed against IgG from a variety of species, including human, mouse, rabbit, rat, chicken, goat, guinea pig, hamster and sheep (Summary of Molecular Probes secondary antibody conjugates—Table 7.1). These anti-IgG antibodies are available with a wide selection of fluorophores, including our:

• Blue-fluorescent Alexa Fluor 350 (), Alexa Fluor 405, Marina Blue, Cascade Blue and Pacific Blue dyes
• Green-fluorescent Alexa Fluor 488 (, , ), Oregon Green 488, Oregon Green 514 (), BODIPY FL () and fluorescein dyes
• Yellow-green–fluorescent Alexa Fluor 430 and Alexa Fluor 514 dyes
• Yellow-fluorescent Alexa Fluor 532 dye ()
• Orange-fluorescent Pacific Orange, Alexa Fluor 546 (), Alexa Fluor 555, tetramethylrhodamine and R-phycoerythrin (R-PE) dyes
• Red-orange–fluorescent Alexa Fluor 568 and Rhodamine Red-X dyes
• Red-fluorescent Alexa Fluor 594 (), Alexa Fluor 610, Texas Red and Texas Red-X dyes
• Far-red–fluorescent Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647 (), Alexa Fluor 660, Alexa Fluor 680 and allophycocyanin dyes
• Infrared-fluorescent Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790 dyes
• Alexa Fluor dye–R-phycoerythrin (R-PE) tandem conjugates, which can each be excited with the 488 nm spectral line of the argon-ion laser, but exhibit long-wavelength emission maxima (627 nm for the Alexa Fluor 610–R-PE conjugates, 667 nm for the Alexa Fluor 647–R-PE conjugates and 702 nm for the Alexa Fluor 680–R-PE conjugates)
• Alexa Fluor dye–allophycocyanin (APC) tandem conjugates, which can each be excited by the 633 nm spectral line of the He-Ne laser or by 635 nm diode lasers with emission beyond 700 nm

Our species-specific anti-IgG antibodies, which are raised against IgG heavy and light chains, are affinity purified and adsorbed against the sera of a number of species to minimize crossreactivity. For multilabeling experiments in which crossreactivity is critical, we offer highly cross-adsorbed goat anti–mouse IgG and goat anti–rabbit IgG antibodies. See the accompanying product information for a complete list of IgG and sera against which our anti-IgG antibodies have been cross-adsorbed.

ReadyProbes Fluorescent Anti-IgG Antibodies

Eight of our bright and photostable Alexa Fluor donkey and goat secondary antibodies are available in ready-to-use dropper bottle formulations:

• Alexa Fluor 488 donkey anti–mouse IgG antibody (R37114)
• Alexa Fluor 594 donkey anti–mouse IgG antibody (R37115)
• Alexa Fluor 488 donkey anti–rabbit IgG antibody (R37118)
• Alexa Fluor 594 donkey anti–rabbit IgG antibody (R37119)
• Alexa Fluor 488 goat anti–mouse IgG antibody (R37120)
• Alexa Fluor 594 goat anti–mouse IgG antibody (R37121)
• Alexa Fluor 488 goat anti–rabbit IgG antibody (R37116)
• Alexa Fluor 594 goat anti–rabbit IgG antibody (R37117)

These ReadyProbes secondary antibodies are prepared from affinity-purified antibodies that react with IgG heavy chains and all classes of immunoglobulin light chains from either mouse or rabbit. ReadyProbes ready-to-use imaging reagents allow you to stain cells without pipetting solutions, making calculations or preparing dilutions. Simply apply 2 drops from the convenient dropper bottles per milliliter of buffer to stain cells before imaging.

Fluorescent Chicken IgY Antibodies

We also offer Alexa Fluor conjugates of chicken IgY secondary antibodies, including anti–mouse IgG, –rabbit IgG, –rat IgG, –human IgG and –goat IgG antibodies. Chicken secondary antibodies have gained popularity because they demonstrate a lower level of nonspecific binding. Chicken IgY antibodies, which are functionally equivalent to mammalian IgG antibodies (and sometimes referred to as chicken IgG antibodies), lack a classical "Fc" domain and are not bound by protein A or protein G, nor do they bind to Fc receptors for mammalian IgG.

Fluorescent Anti-IgM Antibodies

Our anti-IgM conjugates are prepared from well-characterized antibodies that have been purified by IgM affinity chromatography. These anti-IgM conjugates react specifically with IgM heavy chains (µ chains) (Antibody Structure and Classification—Note 7.1). Due to their large size, IgM antibodies do not diffuse well into tissue. In addition, because the IgM µ chain is more highly conserved across different species than are IgG, IgA, or light chains, anti-IgM antibodies may react with IgM from other species.

Isotype-Specific Antibodies

We offer isotype-specific antibodies to aid in multilabeling experiments (Molecular Probes goat anti-mouse isotype-specific antibodies—Table 7.2). The Alexa Fluor goat anti–mouse IgG isotype-specific antibodies have been cross-adsorbed against mouse IgM, IgA, pooled human sera, purified human paraproteins and other isotypes to minimize crossreactivity.

F(ab')₂ Fragments

Our range of anti–mouse IgG, –rabbit IgG,  –rat IgG and –goat IgG antibodies has been expanded to include fluorescent dye–, alkaline phosphatase–, horseradish peroxidase– and biotin-labeled F(ab')₂ fragments (Summary of Molecular Probes secondary antibody conjugates—Table 7.1). These F(ab')₂ fragments are often preferred to whole antibody conjugates because they lack the Fc region (Antibody Structure and Classification—Note 7.1), thereby eliminating nonspecific interactions with Fc receptor–bearing cell membranes and allowing for better penetration into tissue. Please note that the rabbit Fc region may bind nonspecifically to human tissue; consequently, we recommend the F(ab')₂ fragment when using rabbit-derived secondary antibodies on human tissues.

Qdot Secondary Antibody Conjugates

Because of their unique physical and spectroscopic properties, our Qdot secondary antibody conjugates are discussed together with our complete line of Qdot nanocrystal products in Qdot Nanocrystals—Section 6.6 (Qdot nanocrystal secondary antibody conjugates—Table 6.11). The Qdot nanocrystal secondary antibody conjugates exhibit the broad absorption spectra and intense fluorescence output characteristic of Qdot nanocrystals along with the selective binding of F(ab’)₂ fragments, enabling a wide range of immunocytochemical and immunohistochemical applications. Some examples include immunolabeling of GABA_A receptor α1 and γ2 subunits for colocalization analysis in HEK 293 cells and immunodetection of EGFR, E-cadherin and cytokeratin on formalin-fixed, paraffin-embedded (FFPE) tissue array slides.

Enzyme-Labeled and Biotinylated Secondary Antibodies

In addition to fluorescent secondary antibodies, we offer high-activity horseradish peroxidase and alkaline phosphatase conjugates of our species-specific secondary antibodies (Alkaline phosphatase and horseradish peroxidase enzyme conjugates—Table 7.3), as well as several biotin conjugates, for enzyme-based signal amplification techniques. By using an avidin, streptavidin or NeutrAvidin biotin-binding protein bridge, the biotinylated secondary antibodies can be linked to a biotinylated enzyme—a method that is often preferred because it tends to reduce nonspecific staining. Enzyme and hapten conjugates of secondary antibodies are also commonly used in histochemical amplification schemes such as the tyramide signal amplification (TSA) technology and Enzyme-Labeled Fluorescence (ELF) technology, as well as in enzyme-linked immunosorbent assays (ELISAs) (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Phosphatase-Based Signal Amplification Techniques—Section 6.3). The Zenon Alkaline Phosphatase and Horseradish Peroxidase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Zenon Antibody Labeling Kits—Table 7.7) permit the formation of enzyme-labeled antibodies using submicrogram quantities of a primary antibody.

By efficiently blocking nonspecific electrostatic interactions of anionic fluorescent dyes with cationic cell and tissue constituents, the Image-iT FX signal enhancer (I36933) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues, allowing clear visualization of targets that would normally be indistinguishable due to background fluorescence (). Background staining seen with fluorescent conjugates of streptavidin (Fluorescent dyes successfully tested with the Image-iT FX signal enhancer—Table 7.4), goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody is largely eliminated when Image-iT FX signal enhancer is applied to fixed and permeabilized cells prior to staining. Image-iT FX signal enhancer is particularly recommended for use with Alexa Fluor secondary antibodies.

In addition to the stand-alone reagent, we offer the Image-iT FX signal enhancer bundled with our Alexa Fluor goat anti–mouse IgG or Alexa Fluor goat anti–rabbit IgG secondary antibodies (either the standard or highly cross-adsorbed versions) in the Alexa Fluor SFX Kits for immunofluorescence detection:

• Alexa Fluor 488 Goat Anti-Mouse SFX Kit (standard, A31619; highly cross-adsorbed, A31620)
• Alexa Fluor 555 Goat Anti-Mouse SFX Kit (standard, A31621; highly cross-adsorbed, A31622)
• Alexa Fluor 594 Goat Anti-Mouse SFX Kit (standard, A31623; highly cross-adsorbed, A31624)
• Alexa Fluor 647 Goat Anti-Mouse SFX Kit (standard, A31625; highly cross-adsorbed, A31626)
• Alexa Fluor 488 Goat Anti-Rabbit SFX Kit (standard, A31627; highly cross-adsorbed, A31628)
• Alexa Fluor 555 Goat Anti-Rabbit SFX Kit (standard, A31629; highly cross-adsorbed, A31630)
• Alexa Fluor 594 Goat Anti-Rabbit SFX Kit (standard, A31631; highly cross-adsorbed, A31632)
• Alexa Fluor 647 Goat Anti-Rabbit SFX Kit (standard, A31633; highly cross-adsorbed, A31634)

Alexa Fluor Signal Amplification Kits for Mouse Antibodies

The Alexa Fluor Signal Amplification Kits for Mouse Antibodies provide a method for enhancing the detection of mouse primary antibodies using Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 594 dye conjugates, which yield green, red-orange and red fluorescence, respectively. These kits provide two Alexa Fluor antibody conjugates to detect antibodies derived from mouse:

• The Alexa Fluor 488 Signal Amplification Kit (A11054) provides Alexa Fluor 488 rabbit anti–mouse IgG and Alexa Fluor 488 goat anti–rabbit IgG antibodies and is compatible with fluorescein filter sets.
• The Alexa Fluor 568 Signal Amplification Kit (A11066) provides Alexa Fluor 568 rabbit anti–mouse IgG and Alexa Fluor 568 goat anti–rabbit IgG antibodies and is compatible with tetramethylrhodamine filter sets.
• The Alexa Fluor 594 Signal Amplification Kit (A11067) provides Alexa Fluor 594 rabbit anti–mouse IgG and Alexa Fluor 594 goat anti–rabbit IgG antibodies and it compatible with Texas Red dye filter sets.

An Alexa Fluor rabbit anti–mouse IgG antibody conjugate is first used to bind to the mouse-derived primary antibody. The fluorescence is then dramatically enhanced by the addition of an Alexa Fluor goat anti–rabbit IgG antibody (Figure 7.2.4). The Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 594 Signal Amplification Kits for Mouse Antibodies can be used for both fluorescence microscopy and flow cytometry applications and contain sufficient materials for 60–300 assays.

Figure 7.2.4 Antibody amplification scheme provided in the Alexa Fluor 488 Signal Amplification Kits for Mouse Antibodies (A11054). The analogous amplification scheme is used in the Alexa Fluor 568 (A11066) and Alexa Fluor 594 (A11067) Signal Amplification Kits for Mouse Antibodies. An Alexa Fluor rabbit anti–mouse IgG antibody conjugate is first used to bind to the mouse primary antibody. The fluorescence signal is then dramatically enhanced by the addition of an Alexa Fluor goat anti–rabbit IgG antibody.

Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes

The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053) is designed to simultaneously enhance the fluorescence and the photostability of virtually any fluorescein- or Oregon Green dye–containing probe (Figure 7.2.5, Figure 7.2.6). This kit takes advantage of the superior spectral properties of Alexa Fluor 488 conjugates. Alexa Fluor 488 conjugates are considerably brighter and more photostable than fluorescein-labeled probes. In addition, the fluorescence of Alexa Fluor 488 conjugates is not sensitive to pH over a wide pH range, unlike the fluorescence of fluorescein conjugates.

The Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes uses Alexa Fluor 488 conjugates of two different antibodies to amplify the signals from fluorescein-labeled probes. Alexa Fluor 488 anti–fluorescein/Oregon Green dye antibody, which is prepared from a rabbit IgG fraction, is first used to bind to the fluorescein- or Oregon Green dye–labeled target. The fluorescence signal is then dramatically enhanced by addition of the Alexa Fluor 488 goat anti–rabbit IgG antibody. Because the spectra of Alexa Fluor 488 conjugates are remarkably similar to those of fluorescein conjugates (Figure 7.2.7), this kit can be used with optical filters or instrument settings appropriate for fluorescein. The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes can be used for fluorescence microscopy, flow cytometry or other applications that employ fluorescein-conjugated probes; it contains sufficient reagents for 60–120 assays by microscopy or flow cytometry.

Figure 7.2.5 Demonstration of the amplification obtained with the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). Bovine pulmonary artery endothelial cells were labeled with anti–α-tubulin antibody (A11126) in combination with fluorescein goat anti–mouse IgG antibody (F2761) (left panel). The center panel shows the cells after treatment with Alexa Fluor 488 rabbit anti–fluorescein/Oregon Green dye antibody (A11090), and the right panel show the cells after additional labeling with the Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The images were acquired using identical exposure times, and a bandpass filter set appropriate for fluorescein.

Figure 7.2.6 An example of flow cytometry results obtained using the Alexa Fluor 488 Signal Amplification Kit for Fluorescein- and Oregon Green Dye–Conjugated Probes (A11053). Human T-cell leukemia cells (Jurkat) were stained with fluorescein (FITC) mouse anti-CD4 antibody and, as indicated, with Alexa Fluor 488 rabbit anti–fluorescein/Oregon Green dye antibody (A11090) and Alexa Fluor 488 goat anti–rabbit IgG antibody (A11008). The fluorescence values of the negative controls, in which the FITC anti-CD4 antibody was omitted, are shown (black) together with the fluorescence values of the experimental samples (green). The fluorescence values represent the average signals from the population of cells analyzed.

Figure 7.2.7 Absorption and fluorescence emission spectra of fluorescein goat anti–mouse IgG antibody (F2761, (—)) and Alexa Fluor 488 goat anti–mouse IgG antibody (A11001, (- - -)). The fluorescence intensity of the Alexa Fluor 488 conjugate was significantly higher than that of the fluorescein conjugate. The data are normalized to show the spectral similarity.

Amplex ELISA Development Kits for Mouse IgG and for Rabbit IgG

The Amplex ELISA Development Kits for Mouse IgG and for Rabbit IgG (A33851, A33852; TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) provide a comprehensive set of components for creating fluorescence-based ELISAs using horseradish peroxidase conjugates of mouse and rabbit detection antibodies, respectively. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm).

With a high extinction coefficient, good quantum efficiency and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents. In a sandwich ELISA format using C-reactive protein, we can routinely detect 75 pg of antigen using goat anti–mouse IgG antibody and 1 pg using goat anti–rabbit IgG antibody; these detection limits are 25-fold lower than those obtained from the same sandwich ELISA format using the common colorimetric reagent TMB. The Amplex ELISA Development Kits for Mouse IgG and for Rabbit IgG are described in detail in TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2.

Chemiluminescent Alkaline Phosphatase ELISA Kits

We offer four different Chemiluminescent Alkaline Phosphatase ELISA Kits, as well as a Chemiluminescent Alkaline Phosphatase ELISA Sampler Kit. These enzyme-linked immunoassays are based on the CSPD or CDP-Star 1,2-dioxetane substrates for alkaline phosphatase with Sapphire-II or Emerald‑II enhancer in a system designed for rapid and ultrasensitive analyte detection. Each kit provides concentrated assay buffer and blocking reagent, as well as one of the following substrate/enhancer solutions:

• CSPD substrate/Sapphire-II enhancer (in Kit #1, C10552)
• CSPD substrate/Emerald-II enhancer (in Kit #2, C10553)
• CDP-Star substrate/Sapphire-II enhancer (in Kit #3, C10554)
• CDP-Star substrate/Emerald-II enhancer (in Kit #4, C10555)

The Chemiluminescent Alkaline Phosphatase ELISA Sampler Kit (C10556) provides assay buffer, blocking reagent and sample sizes of all four substrate/enhancer solutions. All five kits are described in detail in Phosphatase-Based Signal Amplification Techniques—Section 6.3.

DyeChrome Double Western Blot Stain Kit

The DyeChrome Double Western Blot Stain Kit (D21887) provides a method for the simultaneous trichromatic detection of multiple targets on the same blot using two different enzyme-conjugated secondary antibodies and a general protein stain (Figure 7.2.8). The components of this kit are:

• Horseradish peroxidase (HRP) conjugate of goat anti–rabbit IgG antibody and the Amplex Gold reagent, for yellow-fluorescent detection of a rabbit antibody to a specific protein or proteins
• Alkaline phosphatase conjugate of goat anti–mouse IgG antibody and DDAO phosphate, for far-red–fluorescent detection of a mouse antibody to a specific protein or proteins
• MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone) for blue-fluorescent detection of the total-protein profile
• Appropriate solvents and buffers for the enzymatic reactions
• Detailed protocols (DyeChrome Double Western Blot Stain Kit)

Each DyeChrome Double Western Blot Stain Kit contains sufficient materials to stain approximately 20 minigel blots (6 cm × 9 cm). The two antigens are developed and detected simultaneously, and staining is stable indefinitely on dried blots. The fluorescent signals can be visualized using UV or visible-light illumination.

Figure 7.2.8 A total-protein profile and two specific protein bands visualized on a blot using the DyeChrome Double Western Blot Stain Kit. A twofold dilution series of a protein mixture containing bovine serum albumin (BSA), tubulin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor (from 1 µg to 0.24 ng each) was separated by electrophoresis through a 13% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The DyeChrome Double Western Blot Stain Kit (<a href=">D21887) was used, together with two primary antibodies, to stain all proteins and to visualize two specific proteins. A) The total-protein profile was stained with the blue-fluorescent dye MDPF. B) Tubulin was detected using mouse monoclonal anti–α-tubulin antibody followed by an alkaline phosphatase conjugate of goat anti–mouse IgG antibody, along with DDAO phosphate (red fluorescence). C) BSA was detected using a rabbit IgG antibody against BSA followed by a horseradish peroxidase conjugate of goat anti–rabbit IgG antibody, along with the Amplex Gold reagent (yellow fluorescence). The fluorescent signals were detected separately using appropriate excitation light and emission filters on either the Fluor-S MAX MultiImager documentation system (Bio-Rad Laboratories) or the FLA3000G laser scanner (Fuji Photo Film Co.).

WesternDot Western Blot Kits

The WesternDot 625 Goat Anti-Mouse and Goat Anti-Rabbit Western Blot Kits (W10132, W10142) provide Qdot nanocrystal secondary reagents for the detection of subnanogram amounts of protein on western blots without the complications of time-dependent signal development imposed by enzyme-amplified chemiluminescence detection systems. These kits provide Qdot 625 streptavidin conjugates and biotinylated secondary antibodies for use in combination with user-supplied mouse or rabbit primary antibodies against the protein of interest. In other respects, immunodetection of proteins immobilized on nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes follows standard western blotting protocols. The components of these kits are:

• Biotin-XX goat anti–mouse IgG antibody (in W10132) or biotin-XX goat anti–rabbit IgG antibody (in W10142)
• Qdot 625 streptavidin
• WesternDot blocking buffer
• Wash buffer
• WesternDot staining dish
• Detailed protocols (WesternDot 625 Western Blot Kits)

Each WesternDot 625 Western Blot Kit contains sufficient materials to stain approximately 20 minigel blots (8 cm × 8 cm). Detection of the fluorescence signal can be accomplished using standard UV- or blue light–based gel and blot imaging systems and does not require specialized emission filters.

NANOGOLD and Alexa Fluor FluoroNanogold Conjugates

In collaboration with Nanoprobes, Inc. (www.nanoprobes.com), we offer NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies and streptavidin to facilitate immunoblotting, light microscopy and electron microscopy applications. These reagents include affinity-purified Fab fragments of the goat anti–mouse IgG and anti–rabbit IgG antibodies, as well as of streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates—Table 7.5). Also available is NANOGOLD monomaleimide (N20345, Thiol-Reactive Probes Excited with Visible Light—Section 2.2), which can be conjugated to thiols in the same way that dyes are conjugated to proteins and nucleic acids.

NANOGOLD antibody conjugates are covalently conjugated to the 1.4 nm NANOGOLD gold cluster label, whereas Alexa Fluor FluoroNanogold antibody conjugates are coupled to both a NANOGOLD label and either the Alexa Fluor 488 or Alexa Fluor 594 fluorophore, resulting in gold clusters with green or red fluorescence, respectively. Alexa Fluor FluoroNanogold conjugates have all the advantages of the NANOGOLD cluster, with the additional benefit that they may be used for correlative fluorescence, light and electron microscopy ().

NANOGOLD gold clusters have several advantages over colloidal gold. They develop better with silver than do most gold colloids and therefore provide higher sensitivity. Silver enhancement, such as the system provided in the LI Silver Enhancement Kit (L24919), is described below. Additionally, NANOGOLD particles do not have as high affinity for proteins as do gold colloids, thereby reducing background due to nonspecific binding. Several additional features of NANOGOLD and Alexa Fluor FluoroNanogold conjugates include:

• NANOGOLD gold clusters are an extremely uniform (1.4 nm ± 10% diameter) and stable compound, not a gold colloid.
• NANOGOLD gold clusters are smaller than a complete IgG (H+L) antibody—approximately 1/15 the size of an Fab fragment—and therefore will be able to better penetrate cells and tissues, reaching antigens that are inaccessible to conjugates of larger gold particles.
• NANOGOLD conjugates contain no aggregates, as they are chromatographically purified through gel filtration columns. This feature is in sharp contrast to colloidal gold conjugates, which are usually prepared by centrifugation to remove the largest aggregates and frequently contain significantly smaller aggregates.
• The ratio of NANOGOLD particle to Fab fragment is nearly 1:1, making this product distinct from the 0.2–10 variable stoichiometry of most colloidal gold–antibody preparations. 

NANOGOLD and Alexa Fluor FluoroNanogold products can be used in immunoblotting, light microscopy and electron microscopy (). Standard immunostaining methodologies can be used successfully with NANOGOLD and Alexa Fluor FluoroNanogold immunoreagents. Also, because the concentration of antibody and gold is similar to most commercial preparations of colloidal gold antibodies, similar dilutions and blocking agents are appropriate.

Colloidal Gold Complexes

We also offer Alexa Fluor 488 dye–labeled colloidal gold conjugates, including affinity-purified goat anti–mouse IgG and anti–rabbit IgG antibodies and streptavidin (NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold conjugates—Table 7.5). These conjugates, which have been adsorbed to 5 nm or 10 nm gold colloids, may be used as probes in immunoblotting, light microscopy, fluorescence microscopy or electron microscopy. The fluorescence of these conjugates can be easily detected by standard techniques, but visualization of colloidal gold can be greatly improved using silver-enhancement methods, such as those provided in the LI Silver Enhancement Kit (L24919) described below.

Combining fluorescent secondary detection reagents with colloidal gold to form functional complexes is difficult because the fluorescence of fluorophores such as fluorescein is significantly quenched by proximity to the colloidal gold. We prepare fluorescent colloidal gold complexes with our Alexa Fluor 488 dye, a dye that has superior brightness and photostability. Our Alexa Fluor 488 dye–labeled colloidal gold complexes of anti-IgG antibody and of streptavidin may be used to perform correlated immunofluorescence and electron microscopy in a two-step labeling procedure, rather than in the three-step indirect labeling procedure that is required with conventional nonfluorescent colloidal gold complexes of anti-IgG antibodies or streptavidin.

LI Silver Enhancement Kit

The LI Silver Enhancement Kit (L24919) provides a convenient, light-insensitive silver-enhancement system for use with the NANOGOLD, Alexa Fluor FluoroNanogold and colloidal gold reagents that can be used with electron or light microscopy or for visualization on blots. LI silver is nucleated quickly by NANOGOLD gold clusters or colloidal gold, resulting in the precipitation of metallic silver and the formation of a dark brown to black signal. The system has markedly delayed self-nucleation, resulting in high contrast and very low backgrounds.

Gold particles in the presence of silver (I) ions and a reducing agent such as hydroquinone act as catalysts to reduce silver (I) ions to metallic silver (Figure 7.2.9). The silver is deposited onto the gold, enlarging the particles to between 30 and 100 nm in diameter. Tissues or blots stained with NANOGOLD gold clusters or colloidal gold are "developed" by this autometallographic procedure to give black staining that can be seen in a light microscope. This immunogold silver staining (IGSS) method has been widely used with the NANOGOLD cluster probe. It is one of the most sensitive immunodetection systems available, yielding highly visible, permanent staining with no fading and with detection limits rivaling that of chemiluminescence and radionuclide labeling. Silver-enhanced NANOGOLD staining is compatible with double-labeling techniques, including enzyme-mediated staining. In blots, as little as 0.1 pg of a target IgG antibody can be detected using a NANOGOLD gold cluster labeled with an Fab fragment of a secondary antibody. NANOGOLD streptavidin (N24918, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) has proven to be highly sensitive in detecting biotinylated nucleic acid probes in in situ hybridization studies.

The LI Silver Enhancement Kit (L24919), which provides both an initiator reagent and an enhancer reagent, is compatible with the NANOGOLD and Alexa Fluor FluoroNanogold reagents. Features of the LI silver enhancement method include:

• High-contrast signal for easy light microscope and immunoblot visibility
• Lower background than many other commercial developers
• High sensitivity
• Light-insensitive signal that can be observed under normal room lighting
• Relatively slow development (10–30 minutes) for precise monitoring
• Compatibility with all immunogold reagents

Figure 7.2.9 Mechanism of silver deposition utilized by the LI Silver Enhancement Kit (L24919). Image courtesy of Nanoprobes, Inc.

Protein A and protein G are bacterial proteins that bind with high affinity to the Fc portion of various classes and subclasses of immunoglobulins from a variety of species (Binding profiles of protein A and protein G—Table 7.6). We offer protein A conjugated to several different Alexa Fluor fluorophores:

• Green-fluorescent Alexa Fluor 488 dye (P11047)
• Orange-fluorescent Alexa Fluor 546 dye (P11049)
• Far-red–fluorescent Alexa Fluor 647 dye (P21462)

We also offer protein G conjugated to the Alexa Fluor 488 dye (P11065) and to horseradish peroxidase (P21041).