Choosing and selecting a secondary antibody

We offer a large selection of Invitrogen™ secondary antibodies including fluorescent Invitrogen™ Alexa Fluor™ conjugates, HRP conjugates, and other conjugates for a variety of host and target species. Our extensive selection enables researchers to optimize experiments to obtain excellent results. Various secondary antibody applications require different specificities, sensitivities, and conjugations.

What is a secondary antibody?

A secondary antibody is an antibody designed to target a primary antibody. Secondary antibodies are often used in combination with primary antibodies to detect target proteins in various immunoassays, like western blots, ELISA, and immunofluorescence. Many secondary antibodies are conjugated to other molecules, like Alexa Fluor dyes or horseradish peroxidase (HRP), which enable detection of the secondary antibody.

It is possible to use conjugated primary antibodies, but secondary antibodies provide many advantages. Using a secondary antibody makes it easy to choose a different conjugate no matter the primary antibody. For example, the same primary can be used with a secondary antibody conjugated to HRP in a western blot and with a different secondary antibody conjugated to Alexa Fluor 488 dye in immunofluorescence.

Also, secondary antibodies enhance detection by localizing more conjugate at the antigen than is possible with a labeled primary antibody. By using secondary antibodies, one avoids needing to chemically label (conjugate) primary antibodies, which are more specialized and costly to obtain. Conjugation (which is amino acid-specific) can interfere with the primary antibody’s recognition of the antigen.

Selecting the right secondary antibody

To successfully choose a secondary antibody, one that is best for your application and research, consider the following factors:

  • Host and target species
  • Targeted reactivity
  • Purification
  • Cross-adsorption
  • Multiplexing
  • Antibody class and subclass
  • Whole antibodies vs. fragments
  • Conjugates
  • Biotin-binding proteins

The application, primary antibody, and experimental design often determine the type of secondary antibody needed. Use our antibody search tool to identify the secondary antibody products you need. For more information on all the factors to consider when selecting a secondary antibody, see below.

The host species is the animal in which the secondary antibody was generated. For example, when rabbit IgG is used to immunize a goat, the result is production of goat anti-rabbit secondary antibodies. We offer secondary antibodies generated in chicken, donkey, goat, mouse, rabbit, rat, and sheep.

Keep in mind that the species used to generate the secondary antibody should always be different from the primary antibody host and target species. For instance, if a primary antibody was derived from a rabbit, then one must use an anti-rabbit secondary antibody that was generated in a species other than rabbit.

Most primary antibodies are produced in mouse or rabbit hosts; therefore, anti-mouse IgG and anti-rabbit IgG are the most popular types of secondary antibodies. Goat is the host species most easily and frequently used by manufacturers to produce polyclonal anti-mouse and anti-rabbit secondary antibodies. Consequently, goat secondary antibodies against mouse IgG and rabbit IgG are commercially available in the widest variety of forms. Several kinds of anti-mouse and anti-rabbit secondary antibodies from other host species are also available.

Next, ensure that the secondary antibody will detect the intended primary antibody target. Reactivity includes not only the target species, but also the target immunoglobulin (Ig) class or subclass, and whether the secondary antibody binds to a particular part of the antibody (see more below).

Determining the needed target species is typically straightforward; it is the species from which the primary antibody was derived. If one is using a rabbit polyclonal primary antibody, then an anti-rabbit secondary antibody is needed. If one wants to detect endogenous antibody in a mouse sample, an anti-mouse secondary antibody is required. We offer secondary antibodies against 17 different target species:

Most secondary antibodies are purified from serum obtained from an immunized animal. Affinity purification involves passing the antibody-containing serum through a column packed with porous resin onto which a ligand has been immobilized. The affinity ligand and antibody bind to one another, after which other components of the serum can be washed away before finally eluting and recovering the antibody (now purified) from the column using a buffer that decouples the ligand-antibody interaction. This type of purification can be accomplished using either a ligand that recognizes antibodies (e.g., Protein G) or a ligand that the antibody recognizes (e.g., the target antigen).

Using Protein G as the affinity ligand results in purification of all antibodies (primarily IgG) from serum without regard to their antigen specificity. Using a target antigen as the affinity ligand results in purification of only antigen-specific antibodies (immunoglobulins, any subclasses) from serum.

Cross-adsorption of secondary antibodies is helpful in eliminating cross-reactivity from other nontarget antibodies and proteins, thereby increasing the antibody’s specificity. Cross-adsorption involves passing the affinity-purified secondary antibody over a column containing immobilized antibodies or serum proteins from other species. The secondary antibodies that recognize these other species’ antibodies or serum proteins will bind to the column, whereas those without cross-reactivity to flow through. By increasing the number of different species of serum components in the adsorption column, one can generate antibodies that exhibit cross-reactivity to fewer and fewer species.

For even further elimination of cross-reactivity, try Invitrogen™ Superclonal™ secondary antibodies. Superclonal secondary antibodies represent a breakthrough in recombinant antibody technology. The proprietary screening and production process yields specific mixtures of recombinant goat or rabbit secondary antibodies that bind with the epitope-specific precision of monoclonal antibodies while also achieving the multi-epitope coverage and sensitivity of polyclonal antibodies.

Below is an example of the use of a cross-adsorbed secondary antibody, Goat Anti–Mouse IgG, Alexa Fluor 488 Conjugate (Cat. No. A11029). This secondary antibody has been cross-adsorbed against bovine, goat, rabbit, rat, and human IgG, as well as human serum. The result is very little cross-reactivity and a low background.

Figure 1. BPAE (bovine pulmonary artery endothelial) cells were fixed and permeabilized using the Image-iT Fixation/Permeabilization Kit (Cat. No. R37602), labeled with an anti–beta-tubulin antibody, and stained with an Alexa Fluor 488 Goat Anti-Mouse secondary antibody (Cat. No. A11029) and Click-iT EdU Alexa Fluor 647 (Cat. No. C10340). Images were acquired on a Nikon upright microscope (60x).

For many antibody detection [RG1] applications, multiplexing can be an option. Multiplexing involves using distinct fluorescent dyes to visualize different targets simultaneously. Highly cross-adsorbed antibodies work well in multiplexing because they decrease species cross-reactivity and background. It is important to choose conjugates that don’t have overlapping emission spectra.

Below is an example of multiplexing using goat anti-rabbit IgG Alexa Fluor 488 conjugate (Cat. No. A11034), DAPI, and Alexa Fluor 594 phalloidin.

Figure 2. Immunocytochemistry analysis of HeLa cells stained with Visfatin ABfinity™ Recombinant Rabbit Monoclonal Antibody.
(A)
The primary antibody was detected using goat anti-rabbit IgG Alexa Fluor 488 conjugated secondary antibody (Cat. No. A11034; green). (B) DAPI was used to stain the nucleus (blue). Alexa Fluor 594 phalloidin was used to stain actin (red). (C) Composite image of cells showing cytoplasmic and nuclear localization of visfatin.

There are several different classes of antibodies, each with a particular purpose within the normal immune system. We currently offer secondary antibodies that target IgG, IgM, IgA, IgE, and IgD. Each of these different antibodies has specific heavy chains: gamma, mu, alpha, epsilon, and delta, respectively. IgA and IgM also contain an additional 15 kDa J chain, which is bound to the antibody through disulfide bonds and helps form a functioning multimeric antibody. IgG is further divided into subclasses—IgG1, IgG2, etc.—based on differences in amino acid composition.

It is important to choose a secondary antibody that targets the class or subclass of the primary antibody being used. This is especially important when using a monoclonal primary antibody, because monoclonal antibodies, by definition, belong to only one subclass each.

Secondary antibodies are available in whole antibody and antibody fragment formats. Determining whether to use a whole antibody or a fragment of antibody is often application-dependent. For instance, antibody fragments are best for immunohistochemistry and immunofluorescence because of their smaller size and ability to penetrate tissues. Whole antibodies are the most frequently used secondary antibodies in most other applications.

Whole antibodies, containing heavy and light chains, provide strong divalent binding to the variable regions while providing a sufficient constant region for attachment of signal-generating dyes or enzymes. However, whole antibodies can result in high background and lower specificity because the light chain is shared by all immunoglobulins and increases cross-reactivity.

F(ab’)2 fragments result from digestion of a whole antibody with pepsin. These antibody fragments contain the strong divalent binding found in the variable regions but lack the Fc portion of the antibody. Therefore, F(ab’)2 fragments are often the preferred type of secondary antibody for use with samples where Fc receptors may be present. They are also smaller than whole antibodies and may penetrate some samples better. However, the smaller size also means that fewer dyes or enzymes can be conjugated to the F(ab’)2 fragment, resulting in secondary antibodies that may be less sensitive than whole antibody options. Fab’ fragments result from digestion of a whole antibody with papain. These antibody fragments contain a single binding domain, with a small portion of constant region. Fab’ fragments may be used in specific applications where the bivalent antibody may be less advantageous.

Figure 3. Names and structures of antibody fragments.

For more information visit our Antibody Fragmentation page.

The choice of conjugate depends upon the application and how the secondary antibody is going to be detected. Is this a cell imaging experiment where the brightness and photostability found in the Alexa Fluor dyes is needed? Is the high activity of an HRP conjugate needed for sensitive western blot detection via a chemiluminescent substrate? Below is a description of each of the types of secondary antibody conjugates we offer.

  • Alexa Fluor Conjugates: We offer 18 different Alexa Fluor dyes as secondary antibody conjugates. Alexa Fluor secondary antibodies exhibit superior brightness and photostability and outperform conventional fluorescent secondary antibodies. With more than 50,000 publications citing their use, Alexa Fluor conjugates have defined the last two decades of fluorescent imaging. To learn more visit Alexa Fluor Dyes and Alexa Fluor 488.
  • Alexa Fluor Tandem Conjugates: We offer a number of Streptavidin Phycoerythrin (RPE) and Allophycocyanin (APC) tandem conjugates with Alexa Fluor dyes for use in flow cytometry. The Alexa Fluor conjugates of RPE are more fluorescent than classic dye RPE conjugates, resulting in better sensitivity to capture rare events.
  • Other Premium Fluorescent Conjugates: We’ve created many Molecular Probes™ fluorescent dyes over the years. These specialty dyes provide many options to customers looking for fluorescent dyes. For example, BODIPY™ FL dyes have unique hydrophobic properties and a relatively long excited-state lifetime. Oregon Green™ dye, with a pKa of 4.6, can be used to monitor pH change. Cascade Blue™ dye shows less spectral overlap with green fluorescent dyes.
  • Pacific Conjugates: We offer three Pacific dyes optimized for detection using the 405 nm laser of a flow cytometer. Pacific Blue™, Pacific Green™, and Pacific Orange™ dye conjugates can be simultaneously excited at 405 nm and emit at 455 nm, 500 nm, and 551 nm, respectively, facilitating three-color analysis. Conjugates of these dyes are strongly fluorescent even at neutral pH.
  • Qdot™ Conjugates: Qdot labeled secondary antibodies are bright—as much as 50 times brighter compared to organic dyes—and they do not photobleach, ever. Because all Qdot probes are excited with the same wavelength, but emit at different wavelengths, all 8 colors can potentially be detected at the same time.
  • Classic Fluorescent Conjugates: Traditional fluorescent dyes, like fluorescein (FITC), tetramethylrhodamine (TRITC), and Cy®3, Cy®5, and Texas Red™ dyes, now have Alexa Fluor dye alternatives, which provide better brightness and photostability. However, we still provide secondary antibodies with these conjugates for customers who may be trying to replicate historical experiments. Additionally, there may be applications where these dyes are especially useful, such as taking advantage of the high rate of photobleaching or pH sensitivity of fluorescein.
  • Classic Fluorescent Tandem Conjugates: Most early tandem dyes, like RPE, Texas Red dye, RPE–Cy®5 (which we sell under the trademark name TRI-COLOR™), and RPE–Cy®5.5, have Alexa Fluor alternatives, which provide better performance. However, we still provide secondary antibodies with these conjugates for customers who may be trying to replicate historical experiments. These conjugates are most frequently used in multicolor flow cytometry experiments.
  • R-Phycoerythrin and Allophycocyanin: We offer secondary antibody conjugates of R-phycoerythrin and allophycocyanin dyes. R-phycoerythrin (RPE) and allophycocyanin (APC) are intensely bright phycobiliproteins, isolated from red algae, that exhibit extremely bright fluorescence with high quantum yields. These large protein-based fluorescent molecules are used primarily for flow cytometry, microarray assays, ELISAs, and other applications that require high sensitivity but not photostability.
  • Enzyme Conjugates: We offer both high-activity horseradish peroxidase (HRP) and alkaline phosphatase (AP) secondary antibody conjugates for customers completing western blots, ELISA, immunohistochemistry, or other enzyme-driven applications. Enzyme conjugates are used in conjunction with a colorimetric, fluorogenic, or chemiluminescent substrate to create a detectable signal. Since each molecule of enzyme can convert many molecules of substrate, this is a form of signal amplification controlled by the length of time the reaction is allowed to continue.
  • Biotin Conjugates: We offer a wide variety of biotin and DSB biotin conjugates. Biotin is a small molecule, a hapten, which is bound by avidin, streptavidin, or NeutrAvidin™ protein with high affinity. Utilizing a biotin-conjugated secondary antibody allows one to use the same secondary antibody in multiple applications, simply by switching the streptavidin conjugate being used. Utilizing biotin/streptavidin also enables signal enhancement. A fluorescent secondary antibody has about 5 molecules of dye conjugated to it. In a biotin/streptavidin system, the secondary antibody may have 5 molecules of biotin, each bound to a molecule of streptavidin, each of which has 5 dye molecules attached to it. However, endogenous biotin may cause background in biotin-rich samples. Thus, it may not be the optimal system for every application.
  • Gold Conjugates: We have gold conjugates for customers using secondary antibodies in electron microscopy. We also offer secondary antibodies conjugated with both Alexa Fluor dye and gold, which may be used for correlative fluorescence immunofluorescence and electron microscopy.
  • Agarose and Sepharose™ Resin Conjugates: Secondary antibodies can be used in antibody purification. By conjugating secondary antibodies to a column matrix like beaded agarose (Sepharose is a brand of beaded agarose), antibodies can be purified based on the specificity of the secondary antibody.
  • Unconjugated Secondary Antibodies: In addition to our wide variety of conjugated secondary antibodies, we make available our unconjugated secondary antibodies as well. These are the same high-quality secondary antibodies used to make our industry-leading conjugates.

For applications in which the primary antibody is labeled with a biotin tag, a biotin-binding protein makes a suitable detection reagent. Both avidin and streptavidin bind very strongly to biotin and allow a single detection reagent to be used with multiple primary antibodies, regardless of host.

In addition to antibodies, many other proteins and molecules can be labeled with biotin and used as primary probes for detection with avidin or streptavidin "secondary" probes. If a primary antibody is not commercially available in biotin-labeled form, it can be labeled using one of our EZ-Link™ Biotinylation Reagents or Kits.

We offer three different kinds of biotin-binding proteins, which are described in the following table.

Comparison of available biotin-binding proteins.

  Avidin Streptavidin NeutrAvidin™ Protein
Molecular mass 67 kDa 53 kDa 60 kDa
Biotin-binding sites 4 4 4
Isoelectric point (pI) 10 6.8 to 7.5 6.3
Specificity Low High Highest
Affinity for biotin (Kd) 10-15 M 10-15 M 10-15 M
Nonspecific binding High Low Lowest