Molecular Probes

Section 7.2 — Secondary Immunoreagents

Molecular Probes provides scientists with an extensive and growing selection of secondary immunoreagents for use in fluorescence microscopy, flow cytometry, microplate assays, protein and nucleic acid blots and microarrays and several other techniques. Section 7.1 highlights the dyes we use to prepare several of our most important blue-, green-, orange-, red- and infrared-fluorescent secondary immunoreagents (Table 7.1), including our outstanding Alexa Fluor conjugates (Product Highlight: The Alexa Fluor Dye Series — Peak Performance across the Visible Spectrum). Because of their superior brightness and photostability (Figure 1.9, , , Figure 1.28), the Alexa Fluor conjugates are superior to most conventional fluorescent secondary reagents (Figure 7.36, Figure 7.37, Figure 7.38) and are the detection reagents of choice for many fluorescence-based immunoassays, in situ hybridization and bead-based applications (Product Highlight: Guide to Labeling Antibodies with Alexa Fluor Dyes). Properties of the low molecular weight dyes that we use to prepare our conjugates are described in detail in Chapter 1, and in brief in Section 7.1. In addition to our extensive line of species-specific anti-IgG antibodies, anti-IgM antibodies, isotype-specific antibodies and F(ab')₂ fragments (Technical Focus: Antibody Structure and Classification), we prepare a variety of 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 (see below).

Because of the uniqueness and importance of the reagents, we have separated the discussion of our Zenon antibody labeling technology into a separate section (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 (Figure 7.57) and quantitative (Figure 7.58) labeling of an intact mouse, rabbit, goat or human IgG antibody (Figure 7.56). 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
• Fluorescence colors can be mixed and matched in any combination for multicolor experiments
• The Zenon labeled antibody complexes can be combined with labeled primary and secondary antibodies in most protocols

In many applications, the lower molecular weight of the Zenon labeling reagents also makes them superior to labeled secondary antibodies in their ability to penetrate tissues. The fluorescence intensity of the Zenon labeled antibody complexes, however, may be two- to threefold less than that of a labeled intact secondary antibody.

Species-Specific Secondary Antibodies

Anti-IgG Antibodies

Molecular Probes offers secondary antibody conjugates directed against IgG from a variety of species, including human, mouse, rabbit, rat, chicken, goat, guinea pig, hamster and sheep (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 (, , , ), Alexa Fluor 500, 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 and tetramethylrhodamine dyes
• Red-orange–fluorescent Alexa Fluor 568 and Rhodamine Red-X dyes
• Red-fluorescent Alexa Fluor 594 (), Texas Red and Texas Red-X fluorophores
• Far-red–fluorescent Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647 (), Alexa Fluor 660 and Alexa Fluor 680 dyes
• Infrared-fluorescent Alexa Fluor 700 and Alexa Fluor 750 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 He–Ne laser at 633 nm or by the krypton-ion laser at 647 nm with emission beyond 700 nm
• Red-orange–fluorescent DyeMer 488/605, red-fluorescent DyeMer 488/615 and far-red–fluorescent DyeMer 488/630 dyes, bifluorophores that can each be excited by the 488 nm spectral line of the argon-ion laser and emit at 605, 615 or 630 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 Table 7.2 for a complete list of IgG and sera against which our anti-IgG antibodies have been cross-adsorbed.

Molecular Probes also offers chicken anti-mouse, -rabbit, -rat, -goat and -human secondary antibodies (Table 7.3). 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, 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.

The CMNB-caged fluorescein conjugates of the goat anti–mouse IgG and goat anti–rabbit IgG antibodies (G21061, G21080) permit some unique experimental protocols, including the light-mediated targeted tagging of single cells or a few cells in tissues (Section 5.3) and use as a photoaddressable hapten, in a manner similar to photolithography. These conjugates are essentially colorless and nonfluorescent until illuminated with ultraviolet light, whereupon sites labeled by the caged fluorophore conjugate yield green-fluorescent staining. This photoactivated fluorescence (PAF) can be measured as an increase in signal, even in the presence of a highly autofluorescent background or other green-fluorescent probes. Furthermore, the fluorescein dye that is liberated serves as a hapten that can be specifically detected and the signal amplified by anti-fluorescein/Oregon Green antibody conjugates (Section 7.4, Figure 7.71).

We offer the high-activity horseradish peroxidase and alkaline phosphatase conjugates of goat anti–mouse IgG, rabbit anti–mouse IgG, goat anti–rabbit IgG and rabbit anti–goat IgG antibodies (Table 7.4), as well as biotin conjugates of goat anti–mouse IgG antibody and of the F(ab')₂ fragments of goat anti–mouse IgG antibody and goat anti–rabbit IgG antibody (B2763, B11027, B21078). By using an avidin, streptavidin or NeutrAvidin biotin-binding protein bridge, scientists can link our biotinylated or DSB-X biotin–labeled secondary antibodies to a biotinylated enzyme — a method that is often preferred because it tends to reduce nonspecific staining. Links to avidin and streptavidin conjugates made through DSB-X biotin are readily reversible (see Section 7.6 for a description of our unique DSB-X biotin technology). Enzyme and hapten conjugates of secondary antibodies are also commonly used in histochemical amplification schemes such as the tyramide signal amplification (TSA) technology (Section 6.2) and Enzyme-Labeled Fluorescence (ELF) technology (Section 6.3, ). Our Zenon Alkaline Phosphatase and Horseradish Peroxidase Antibody Labeling Kits (Section 7.3, Table 7.14) permit the formation of enzyme-labeled antibodies using as little as submicrogram quantities of a primary antibody. Enzyme-conjugated antibodies are also used in a wide variety of ELISA methods, such as in our Amplex Red ELISA Kits (see below).

Anti-IgM Antibodies

In response to requests from researchers wanting to apply Alexa Fluor dye technology to the detection of IgM monoclonal antibodies, we have added goat anti–mouse IgM, goat anti–rat IgM and goat anti–human IgM antibodies to the list of antibodies we offer as conjugates of our Alexa Fluor dyes (Table 7.1). The anti-IgM conjugates are prepared from well-characterized antibodies that have been purified by IgM affinity chromatography and react specifically with IgM heavy chains (µ chains) (Technical Focus: Antibody Structure and Classification). To minimize crossreactivity, the goat anti–mouse IgM antibodies have been adsorbed against human IgG₁, IgG_2a, IgG_2b, IgG₃, IgA, human serum and purified human paraproteins. The goat anti–human IgM antibodies have been adsorbed against human IgG and IgA. Due to their large size, IgM antibodies do not diffuse well into tissue, and 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

Molecular Probes offers isotype-specific antibodies to aid in multilabeling experiments (Table 7.5). 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 goat anti–mouse IgG and goat anti–rabbit IgG antibodies has been expanded to include fluorescent dye–, alkaline phosphatase–, horseradish peroxidase– and biotin-labeled F(ab')₂ fragments (Table 7.1). These F(ab')₂ fragments are often preferred to whole antibody conjugates because they lack the Fc region (Technical Focus: Antibody Structure and Classification), 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, Molecular Probes recommends the F(ab')₂ fragment when using rabbit-derived secondary antibodies on human tissues.

Image-iT FX Kits — All-in-One Kits for Fluorescence Imaging of Fixed Cells

Image-iT FX Kits

The Image-iT FX Kits (Table 7.6) provide some of our best secondary detection reagents and the supporting materials needed for optimal imaging of fixed cells and tissue sections:

• Alexa Fluor conjugates of goat anti–mouse IgG antibody, goat anti–rabbit IgG antibody or streptavidin deliver superior photostability and brightness (Table 7.7)
• ProLong Gold antifade reagent reduces photobleaching (Figure 23.25, , ; see Section 23.1 for more details)
• Image-iT FX signal enhancer improves the signal-to-noise ratio (, , )
• A sample pack of two CultureWell chambered coverglasses makes sample processing more convenient (Figure 23.35, see Section 23.3 for more details)

Each Image-iT FX Kit provides sufficient materials to perform 50–100 assays. Furthermore, the components of each kit are available separately (Alexa Fluor secondary antibodies, Table 7.1; Alexa Fluor streptavidins, Section 7.6, Table 7.23; ProLong Gold antifade reagent, P36930, Section 23.1; Image-iT FX signal enhancer, I36933; CultureWell chambered coverglasses, C37000, C37005; Section 23.3) for flexibility in experimental design.

Image-iT FX Signal Enhancer

By efficiently blocking nonspecific interactions of a wide variety of fluorescent dyes with 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 (Table 7.8), 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 may also effectively prevent nonspecific staining that is typically blocked with 1–2% BSA or 10% serum treatment, in some cases eliminating the need for another step in the staining protocol.

Alexa Fluor Signal Amplification Kits

Molecular Probes' Alexa Fluor Signal Amplification Kits are designed to substantially increase the signals obtained by immunofluorescence techniques, thus permitting detection of low-abundance targets (Figure 7.46, Figure 7.47). Each kit takes advantage of the superior brightness and photostability of Alexa Fluor antibody conjugates. The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes (A11053) dramatically enhances the fluorescence and photostability of virtually any fluoresceinated probe. The three kits for mouse antibodies (Table 7.9) can be used to sensitively detect mouse primary antibodies. All of the Alexa Fluor Signal Amplification Kits contain detailed protocols for staining adherent cells grown on coverslips. The kits also contain protocols for use in flow cytometry.

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, Alexa Fluor(R) 488 Signal-Amplification Kit for Fluorescein and Oregon Green(R) Dye–Conjugated Probes) is designed to simultaneously enhance the fluorescence and the photostability of virtually any fluorescein- or Oregon Green dye–containing probe (Figure 7.46, Figure 7.47). This kit takes advantage of the superior properties of Alexa Fluor 488 conjugates. Alexa Fluor 488 conjugates are considerably brighter and more photostable than fluorescein-labeled probes (Figure 1.9, , ). 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 (Figure 1.12).

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 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 7.48), the kit can be used with optical filters or instrument settings appropriate for fluorescein (Section 23.5, Table 23.11). The Alexa Fluor 488 Signal Amplification Kit for Fluorescein-Conjugated Probes can be used for fluorescence microscopy, flow cytometry, blots, microarrays and probably any other application that uses fluorescein-conjugated probes; it contains sufficient reagents for 60–120 assays by microscopy or flow cytometry.

Alexa Fluor Signal Amplification Kits for Mouse Antibodies

Molecular Probes offers three Alexa Fluor Signal Amplification Kits for Mouse Antibodies, permitting enhanced detection of mouse primary antibodies using conjugates of our superior Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 594 dyes, which yield green, red-orange and red fluorescence, respectively (Table 7.9). These kits each use two Alexa Fluor conjugates to detect antibodies derived from mouse. 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 conjugate of a goat anti–rabbit IgG antibody (Figure 7.49). The Alexa Fluor 488 (Alexa Fluor(R) 488 Signal-Amplification Kit for Mouse Antibodies), Alexa Fluor 568 (Alexa Fluor(R) 568 Signal-Amplification Kit for Mouse Antibodies) and Alexa Fluor 594 (Alexa Fluor(R) 594 Signal-Amplification Kit for Mouse Antibodies) Signal Amplification Kits for Mouse Antibodies can be used for both fluorescence microscopy and flow cytometry and contain sufficient materials for 60–300 assays.

SensiFlex ELISA Development Kits

The SensiFlex ELISA Development Kits (S33853, S33854) offer an extremely sensitive fluorometric detection method for β-lactamase–amplified enzyme-linked immunosorbent assays (ELISAs). The Fluorocillin Green 495/525 reagent provided in these kits is a novel substrate and, following cleavage by β-lactamase, emits a green-fluorescent signal (emission maxima ~525 nm). Fluorocillin Green reagent has a broad dynamic range of fluorescence signal, is more sensitive than common colorimetric substrates and displays only modest hydrolysis when incubated for extended periods from pH 5.5 to 8.0. Additionally, Fluorocillin Green reagent effectively reports β-lactamase activity in the presence of EDTA, many detergents, salts and sodium azide. Developing ELISA protocols that maximize both dynamic range and limit of detection involves optimizing the concentrations of both the enzyme and substrate. The robust enzyme kinetics of the reaction between β-lactamase and Fluorocillin Green reagent are consistent, even in the face of changes in enzyme and substrate concentration. This feature makes assay development using β-lactamase and Fluorocillin Green reagent easier than for assays that employ an enzyme with faster kinetics (e.g., horseradish peroxidase). The SensiFlex ELISA Development Kits contain:

• Fluorocillin Green 495/525 β-lactamase substrate
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS)
• β-Lactamase TEM-1 conjugate of goat anti–mouse IgG antibody (in Kit S33853) or anti–rabbit IgG antibody (in Kit S33854)
• Fluorocillin stop reagent
• Sodium bicarbonate buffer, pH ~9.3
• Bovine serum albumin (BSA)
• Tween 20
• Nunc-Immuno MaxiSorp U96 plate
• Detailed protocols (SensiFlex ELISA Development Kit with Fluorocillin Green Reagent)

Sufficient reagents are provided in each kit for performing approximately 500 assays using 100 µL reaction volumes. Fluorocillin Green 495/525 β-lactamase substrate (F33952), as well as the β-lactamase TEM-1 conjugates of the goat anti–mouse IgG and anti–rabbit IgG secondary antibodies (G31567, G31568), are also available from Molecular Probes as standalone products.

Amplex Red and Amplex UltraRed ELISA Kits

Amplex Red ELISA Kits

Molecular Probes' Amplex Red ELISA Kits offer an extremely sensitive fluorometric or colorimetric detection method for horseradish peroxidase (HRP)–amplified enzyme-linked immunosorbent assays (ELISAs). The Amplex Red ELISA Kit #1 (A22170) contains an HRP goat anti–mouse IgG antibody conjugate, which can be used for the ELISA detection of any mouse IgG antibody. The Amplex Red ELISA Kit #2 (A22171) contains the versatile protein G conjugate of HRP, which can be used for the ELISA detection of IgGs from most commonly used species, including human, mouse, rabbit, goat, sheep, bovine and horse. The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, ) provided in these kits is a highly sensitive and stable probe for the detection of HRP activity. In the presence of HRP, the Amplex Red reagent reacts with hydrogen peroxide with a 1:1 stoichiometry to form the fluorescent product resorufin (R363, Section 10.1; absorption/emission maxima ~571/585 nm, Figure 10.59). Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red ELISA Kit #1 with the HRP–goat anti–mouse IgG antibody conjugate has detection limits of as little as 10 pg/microplate well of a mouse IgG by fluorometry or 50 pg/microplate well by colorimetry (Figure 7.50). The Amplex Red ELISA Kit #2 with HRP–protein G has detection limits of as little as 1 ng/microplate well of a mouse IgG by fluorometry or 3 ng/microplate well by colorimetry (Figure 7.51) in 96-well plates.

Each Amplex Red ELISA Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Concentrated reaction buffer
• Hydrogen peroxide (H₂O₂)
• Horseradish peroxidase (HRP) conjugate of goat anti–mouse IgG antibody (in Kit #1, A22170) or of protein G (in Kit #2, A22171)
• Detailed ELISA protocols (Amplex(R) Red ELISA Kit #1, with Goat Anti–Mouse IgG, Horseradish Peroxidase Conjugate, Amplex(R) Red ELISA Kit #2, with Protein G, Horseradish Peroxidase Conjugate)

Each kit provides sufficient reagents for approximately 1000 ELISAs using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. Our HRP conjugates of the goat anti–mouse IgG antibody (G21040), goat anti–rabbit IgG antibody (G21234) and protein G (P21041) are available separately. HRP conjugates of additional antibodies that can be used with the Amplex Red reagent (A12222, A22177; Section 10.5) are listed in Table 7.4.

Amplex ELISA Development Kits for Rabbit and Mouse IgG

The Amplex ELISA Development Kits for Mouse IgG (A33851) and for Rabbit IgG (A33852) provide a comprehensive set of components for creating a fluorescence-based ELISA using a mouse or rabbit primary antibody, 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) (). Because the Amplex UltraRed peroxidation product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples. 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, Molecular Probes scientists are routinely able to 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. Each Amplex ELISA Development Kit contains:

• Amplex UltraRed reagent
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS), pH 7.2
• Horseradish peroxidase conjugate of goat anti–mouse IgG antibody (in A33851) or goat anti—rabbit IgG antibody (in A33852)
• Amplex stop reagent
• Hydrogen peroxide (H₂O₂)
• 0.1 M sodium bicarbonate buffer, pH ~9.3
• Bovine serum albumin (BSA)
• Tween 20
• Nunc-Immuno MaxiSorp U96 plate
• Detailed protocols (Amplex(R) ELISA Development Kit for Mouse IgG with Amplex(R) UltraRed Reagent, Amplex(R) ELISA Development Kit for Rabbit IgG with Amplex(R) UltraRed Reagent)

Sufficient reagents are provided in each kit for 500 microplate assays in a 96-well fluorescence microplate format (100 µL per assay).

Gold-Labeled Immunoreagents

NANOGOLD and Alexa Fluor FluoroNanogold Conjugates

In collaboration with Nanoprobes, Inc. (http://www.nanoprobes.com/), Molecular Probes offers NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies and streptavidin to facilitate immunoblotting, light microscopy and electron microscopy applications (NANOGOLD(R) and FluoroNanogold Conjugates). These reagents include affinity-purified Fab fragments of the goat anti–mouse IgG, goat anti–rabbit IgG and rabbit anti–goat IgG antibodies, as well as of streptavidin (Section 7.6, Table 7.10). Also available are NANOGOLD mono(sulfosuccinimidyl ester) (N20130, Section 1.6, NANOGOLD(R) Mono(sulfosuccinimidyl ester)) and NANOGOLD monomaleimide (N20345, Section 2.2, NANOGOLD(R) Monomaleimide), which can be conjugated to amines (Figure 1.89) and thiols (Figure 2.22), respectively, in the same way that dyes are conjugated to proteins and nucleic acids. NANOGOLD conjugates are covalently conjugated to the 1.4 nm NANOGOLD gold cluster label, whereas Alexa Fluor FluoroNanogold 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 as a result, provide higher sensitivity. Additionally, NANOGOLD particles do not have as high affinity for proteins as do gold colloids, thereby reducing any background due to nonspecific binding. Several additional advantages of NANOGOLD and Alexa Fluor FluoroNanogold streptavidin over colloidal gold conjugates include:

• The 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 absolutely 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 F(ab) is nearly 1:1, making this product distinct from the 0.2–10 variable stoichiometry of most colloidal gold–antibody preparations.
• NANOGOLD cluster–stained targets develop better with silver than do most gold colloids, resulting in higher sensitivity. Silver enhancement, such as the system provided in the LI Silver Enhancement Kit (L24919), is described below.

NANOGOLD and Alexa Fluor FluoroNanogold products can be used in immunoblotting, light microscopy, and electron microscopy to provide clear visibility (). 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

Molecular Probes offers Alexa Fluor 488 dye–labeled colloidal gold conjugates, including affinity-purified goat anti–mouse IgG and goat anti–rabbit IgG antibodies and streptavidin (Table 7.10). 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 we provide 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. Molecular Probes makes 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 for electron or light microscopy or to visualize gold particles 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 7.54). 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 method — known as immunogold silver staining (IGSS) — has been widely used with the NANOGOLD cluster probe; it is one of the most sensitive immunodetection systems available and gives highly visible, permanent staining with no fading, 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, Section 7.6) has proved to be highly sensitive in detecting biotinylated nucleic acid probes in in situ hybridization studies.

The LI Silver Enhancement Kit (L24919, LI Silver Enhancement Kit), which is useful for light microscopy, gels and Western blots, is ideal for use with the NANOGOLD and Alexa Fluor FluoroNanogold reagents and for enhancing colloidal gold products. The advantages of LI silver enhancement include:

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

DAB Histochemistry Kits

The use of horseradish peroxidase (HRP) for enzyme-amplified immunodetection — commonly referred to as immunoperoxidase labeling — is a well-established standard histochemical technique. The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187, Section 7.6). Each kit contains:

• Diaminobenzidine (DAB)
• HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
• H₂O₂ reaction additive
• Blocking reagent
• Staining buffer
• A detailed staining protocol (Diaminobenzidine Histochemistry Kits)

Each kit provides sufficient materials to stain approximately 200 slides.

Captivate Ferrofluid Conjugates and DSB-X Biotin Conjugate Applications

Magnetic separation is a quick, simple technique for isolating cells, organelles, proteins and nucleic acids from complex mixtures, based on bioaffinity principles. The sample containing a target ligand to be isolated is incubated with the superparamagnetic particles coupled with the appropriate affinity reagent, e.g., streptavidin, an antibody or a complementary nucleic acid. The bound target ligand is then separated efficiently from the mixture using a high field-strength magnet.

Molecular Probes, in association with Immunicon Corporation, offers Captivate ferrofluid conjugates of goat anti–mouse IgG antibody (C21473), goat anti–rabbit IgG antibody (C21474) and streptavidin (C21476). Ferrofluids are superparamagnetic particles ~200 nm in diameter that respond to a magnetic field but completely demagnetize when the field is removed. The key feature of the Captivate ferrofluid is its small and relatively uniform particle size, which results in efficient diffusion of the ferrofluid conjugate and rapid kinetics of the binding reaction. Once added to the sample, however, no further mixing is required. Furthermore, the ferrofluid conjugates exhibit significantly higher ligand-binding capacities per mass, as compared with larger-diameter superparamagnetic particles from other suppliers. We also offer the Captivate microscope-mounted magnetic yoke assembly (C24700) and disposable sample chambers (C24701), which have been specially designed for use with the Captivate ferrofluid conjugates (Figure 7.55), for optimum capture of Captivate ferrofluid–tagged cells from such diverse liquid samples as culture media, blood and biological buffers. The Captivate microscope-mounted magnetic yoke assembly includes one free set of