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Selection Guide: Secondary Antibodies and Detection Reagents |
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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.
Contents:
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Overview of Detection Probes
Overview of Western Blotting
Overview of ELISA
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Polyclonal anti-Mouse IgG and anti-Rabbit IgG Secondary Antibodies and Conjugates
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.
To find secondary antibodies with specificity to species other than mouse or rabbit, additional secondary antibodies and various conjugates of each are available using the menu to the right.
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Secondary Antibodies and Conjugates...
Anti-Chicken Antibodies
Anti-Goat Antibodies
Anti-Hamster Antibodies
Anti-Horse Antibodies
Anti-Human Antibodies
Anti-Rat Antibodies
Anti-Sheep Antibodies |
| Directory of product pages for anti-mouse and anti-rabbit secondary antibodies. Products are organized on separate product pages as delineated 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 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 ) |
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Standard-Purity
(goat host; H+L)
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Highly Cross-Adsorbed
(goat host; H+L) |
| Mouse |
Unconjugated
AP, HRP
FITC, TRITC, etc.
Biotin |
405, 488, 549,
594, 633, 649,
680, 750, 800 |
405, 488, 549,
594, 633, 649,
680 |
Pre-diluted
(10 µg/ml) |
Poly-HRP |
| Rabbit |
Unconjugated
AP, HRP
FITC, TRITC, etc.
Biotin |
405, 488, 549,
594, 633, 649,
680, 750, 800 |
405, 488, 549,
594, 633, 649,
680 |
Pre-diluted
(10 µg/ml) |
Poly-HRP |
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Notes:
- Common Formats includes typical non-conjugated and conjugated antibodies in a variety of forms (e.g., host species, fragment specificities and 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.
- Specialty HRP Conjugates includes stablized (Pre-diluted) horseradish peroxidase (HRP) conjugates for greater convenience, as well as Poly-HRP Conjugates that provide exceptionally high signal especially for immunohistochemical methods.
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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. To view biotinylation products, use the links to the right.
We offer three different kinds of biotin-binding proteins:
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Comparison of available biotin-binding proteins.
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Avidin
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Streptavidin
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NeutrAvidin Protein
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Molecular Weight
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67K
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53K
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60K
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Biotin-binding Sites
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4
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4
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4
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Isoelectric Point (pI)
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10
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6.8-7.5
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6.3
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Specificity
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Low
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High
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Highest
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Affinity for Biotin (Kd)
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10-15 M
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10-15 M
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10-15 M
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Nonspecific Binding
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High
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Low
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Lowest
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View Products...
EZ-Link Biotinylation Reagents
Biotin 3' End DNA Labeling Kit |
| Directory of product pages for biotin detection reagents. Products are organized on separate product pages as delineated 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 |
N/A |
NeutrAvidin
Protein |
Unconjugated
AP, HRP
FITC |
405, 488, 549,
594, 633, 649,
680, 800 |
Pre-diluted
(10 µg/ml) |
High Sensitivity |
Poly-HRP |
| Streptavidin (recombinant) |
Unconjugated
AP, HRP
FITC, TRITC, etc. |
405, 488, 549,
594, 633, 649,
680, 750, 800 |
Pre-diluted
(10 µg/ml) |
High Sensitivity |
Poly-HRP |
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Notes:
- Common Formats includes 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 includes stablized (Pre-diluted) horseradish peroxidase (HRP) conjugates for greater convenience, High Sensitivity Conjugates that are prepared and purified to minimize background and variability, and Poly-HRP Conjugates that provide exceptionally high signal especially for immunohistochemical methods.
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Antibody-Binding Proteins
The antibody-binding proteins Protein A, Protein G and Protein L are useful for preparation of the purified 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 (47 kDa and 22 kDa, 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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Download documents...
Tech Tip#34: Binding characteristics of Protein A, Protein G, Protein A/G and Protein L.
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Specialized Detection Probes and Kits
In particular 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.
Clean-Blot IP Detection Reagent
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Download documents
Tech Tip #64: Immunoprecipitation (IP) technical guide and protocols |
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