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Secondary Antibodies Selection Guide

Choose an appropriate secondary detection probe for Western blots and ELISA.

Thermo Scientific Pierce Secondary Antibodies and Detection Reagents are available in a variety of formats and conjugate types. Although these many options provide for excellent performance in many kinds of antibody-based detection and assay techniques, they also present researchers with a seemingly overwhelming array of choices. When choosing a secondary antibody product, consideration must be given to species and immunoglobulin specificity, conjugate type, fragment and chain specificity, level of cross-reactivity, and host-species source and fragment composition. This page provides a concise overview of the main classes of secondary probes and a directory of links to relevant product pages.

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Protein Methods Library Home


Overview of Detection Probes

Overview of Western Blotting

Overview of ELISA

 

Polyclonal Secondary Antibodies

Anti-Mouse (GAM) and Anti-Rabbit (GAR) IgG

Most primary antibodies are produced in mouse or rabbit host species; therefore, anti-mouse IgG and anti-rabbit IgG are the most popular classes 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. See the table below for help locating secondary antibodies with specificity for mouse or rabbit antibodies and their conjugation options.

Secondary antibodies with specificity to species other than mouse or rabbit and various conjugates of each are available using the links to the right.

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Secondary Antibodies
(this site - piercenet.com)

Secondary Antibodies
(www.pierce-antibodies.com)
Directory of product pages for anti-mouse and anti-rabbit secondary antibodies. Products are organized on separate product pages as indicated in this table. The "Common Formats" column links to a product page that includes typical non-conjugated and conjugated antibodies in a variety of forms (e.g., host species, fragment specificities and varying cross-adsorption levels). The remaining columns link to product pages that each feature a different specialized class or form of polyclonal goat secondary antibodies. See Notes section for more details.
Specificity Common
Formats
(see Notes)
DyLight Fluor Conjugates Specialty HRP Conjugates
(goat host; H+L )
Standard Purity
(goat host; H+L)
Highly Cross-Adsorbed
(goat host; H+L)
Mouse Unconjugated
AP, HRP
FITC, TRITC, etc.
Biotin
405, 488, 550, 594, 
633, 650, 680, 800
405, 488, 
594, 633, 680
Pre-diluted
(10µg/mL)
Poly-HRP
Rabbit Unconjugated
AP, HRP
FITC, TRITC, etc.
Biotin
405, 488, 550, 594, 
633, 650, 680, 800
405, 488, 
594, 633, 680
Pre-diluted
(10µg/mL)
Poly-HRP

Notes:

  • Common Formats include typical non-conjugated and conjugated antibodies in a variety of forms (e.g., host species, fragment specificities and varying cross-adsorption levels).
  • DyLight Fluor Conjugates are polyclonal goat antibodies against heavy and light chain (H+L) fragments that are conjugated to high-performance Thermo Scientific DyLight Fluorescent Dyes. One product page lists antibodies that are not pre-adsorbed to remove potential species cross-reactivities (Standard Purity), while the second page lists the DyLight Fluor Conjugates that have been Highly Cross-Adsorbed to remove these cross-reactive antibodies.
  • Specialty HRP Conjugates include stabilized (Pre-diluted) horseradish peroxidase (HRP) conjugates for greater convenience as well as Poly-HRP Conjugates that provide exceptionally high signal, especially for immunohistochemical methods.

Secondary Antibodies for Other Species of IgG (at Pierce-Antibodies.com)

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Secondary Antibodies
(this site - piercenet.com)

Secondary Antibodies
(www.pierce-antibodies.com)

Biotin-Binding Proteins

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:

Comparison of available biotin-binding proteins.
  Avidin Streptavidin NeutrAvidin
Protein
Molecular Weight 67K 53K 60K
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) 10-15M 10-15M 10-15M
Nonspecific Binding High Low Lowest

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Biotin-binding Proteins and Conjugates

 

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Overview of Avidin-Biotin Probes

 

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EZ-Link Biotinylation Reagents

Biotin 3' End DNA Labeling Kit

Biotin-binding Affinity Resins

Directory of product pages for biotin detection reagents. Products are organized on separate product pages as indicated in this table. The "Common Formats" column links to a product page that includes typical non-conjugated and conjugated forms of each biotin-binding protein. The remaining columns link to product pages that each feature a different specialized class or form conjugate. See Notes section for more details.
Protein Common Formats DyLight Fluor Conjugates Specialty HRP Conjugates
Avidin Unconjugated
AP, HRP
FITC
N/A N/A N/A
NeutrAvidin
Protein
Unconjugated
AP, HRP
FITC
405, 488, 594, 
633, 680, 800
High Sensitivity N/A
Streptavidin
(recombinant)
Unconjugated
AP, HRP
FITC, TRITC, etc.
405, 488, 594, 
633, 680, 800
High Sensitivity Poly-HRP

Notes:

  • Common Formats include typical non-conjugated and conjugated forms of each protein.
  • DyLight Fluor Conjugates are labeled with high-performance Thermo Scientific DyLight Fluorescent Dyes.
  • Specialty HRP Conjugates include High Sensitivity HRP Conjugates, which are prepared and purified to minimize background and variability, and Poly-HRP Conjugates, which provide exceptionally high signal, especially for immunohistochemical methods.

Antibody-Binding Proteins

The antibody-binding proteins Protein A, Protein G and Protein L are useful for purifying IgG from serum and cell culture supernatant. However, these protein can also be useful as detection probes for ELISA, Western blotting and immunohistochemical staining. Because of their smaller size, Protein A and Protein G (47kDa and 22kDa, respectively) are better able to penetrate into tissue than secondary antibodies. Also, because each antibody binding protein has affinity for the IgG of several species, a single detection probe may be used for multiple primary antibodies.

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Antibody-binding Proteins

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Tech Tip#34: Binding characteristics of Protein A, Protein G, Protein A/G and Protein L

Specialized Detection Probes and Kits

In some methods, typical secondary antibodies are either too specific (e.g., recognize only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof). In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary probe. For example, biotin-binding proteins can be used to detect any species of primary antibody that has been labeled with a biotin tag. However, for certain applications, it is difficult and time-consuming to choose and optimize the components to obtain desired results.

We continue to develop optimized reagents to address the particular requirements of specific detection applications. These specialized detection probes are not easily classified in any of the categories discussed above. For example, the Thermo Scientific Clean-Blot IP Detection Reagent provides specific Western blot detection of immunoprecipitation products without the usual cross-reaction with IP antibody fragments.

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Clean-Blot IP Detection Reagent

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Overview of Immunoprecipitation (IP) Methods

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