Confocal micrograph of the cytoskeleton of a mixed population of granule neurons and glial cells. The F-actin was stained with red-fluorescent Texas Red™-X phalloidin (Cat. No. T7471). The microtubules were detected with a mouse monoclonal anti–ß-tubulin primary antibody and subsequently visualized with the green-fluorescent Alexa Fluor™ 488 Goat Anti–Mouse IgG antibody (Cat. No. A-11001). The image was contributed by Jonathan Zmuda, Immunomatrix, Inc.
Anti-Mouse Secondary Antibodies
Invitrogen™ anti-mouse secondary antibodies are affinity-purified polyclonal antibodies with well-characterized specificity for mouse immunoglobulin classes, subclasses, and fragments. They are useful in the detection, sorting, or purification of the specified target (primary antibody). We offer anti-mouse secondary antibodies produced in various host species, including goat, donkey, rabbit, chicken, rat, cow, and sheep.
- Unlabeled, unconjugated
- Enzyme conjugates (alkaline phosphatase (AP) or horseradish peroxidase (HRP))
- Biotinylated (biotin conjugates)
- Fluorescent conjugates (Alexa Fluor™, fluorescein (FITC), and other classic fluorescent conjugates)
Production of anti-mouse secondary antibodies
Anti-mouse secondary antibodies are generated by immunizing a host animal (e.g., a goat) with a pooled population of immunoglobulins (Ig) from the target species (i.e., mice). After the host animal’s immune system responds to produce anti-mouse Ig antibodies, serum is collected, and the target antibodies are purified. Using different methods of production and purification, we are able to manufacture and offer many different varieties of anti-mouse secondary antibodies, helping researchers to optimize their detection protocols. For example, because mice produce immunoglobulins (Ig) of various classes and subclasses (e.g., IgG1, IgG2, IgM), multiple mouse monoclonal primary antibodies used in a given experiment may have different subclass identities; by using subclass-specific secondary antibodies, these different primary antibodies can be detected simultaneously in multiplex assays, despite being from the same host species (mouse).
Secondary antibodies are typically affinity purified using the target antigen (the primary antibody class, subclass, and fragment) coupled to an agarose support. This affinity purification (positive selection) eliminates nonspecific antibodies and other proteins from the source serum, resulting in a preparation of target-specific polyclonal antibodies that provide high specificity and a low background signal for many applications. Conjugated antibodies are affinity purified before labeling.
Select secondary antibodies are further purified by passing the antibody to over an affinity column containing immobilized serum proteins or IgG of selected species. Any antibodies in the polyclonal preparation that bind (adsorb) to these immobilized serum proteins are thus removed (negative selection). These antibodies are designated as cross-adsorbed or highly cross-adsorbed, based on the amount of processing which has occurred. The additional processing increases the antibody’s specificity and helps to eliminate cross-reactivity from other non-target antibodies and proteins.
Anti-mouse IgG antibodies can also cross-react with proteins from other, non-target species. This includes a possible cross-reactivity to other IgG molecules. Cross-reactivity leads to nonspecified binding of the secondary antibody resulting in decreased sensitivity and high background.
The mouse proteome is homologous to multiple mammalian species’ proteomes (see table). The similarity between proteomes makes the purification process especially important. The cross-reactivity that may occur between species with similar IgG classes (or other proteins) could result in less specific results and high background. Taking extra steps to purify secondary antibodies, and eliminate cross-reactivity results in better experimental outcomes.
Percent total proteome homology between various species and mouse.
|Cat. No.||Invitrogen™ Antibody||Size|
|A-11001||Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate||500 µL|
|A-11029||Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate||500 µL|
|A-21202||Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate||500 µL|
|A-11017||F(ab')2-Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate||250 µL|
|A-11005||Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate||500 µL|
|A-31571||Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate||500 µL|
|A-21121||Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate||250 µL|
|A-31570||Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate||500 µL|
|A-21235||Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate||500 µL|
Detection of beta tubulin in BPAE cells using Alexa Fluor 488 anti-mouse secondary antibody. Cells were fixed and permeabilized using the Image-iT™ Fixation/Permeabilization Kit (R37602), labeled with an anti–beta tubulin antibody, and stained with an Alexa Fluor™ 488 Goat Anti-Mouse secondary antibody (Cat. No. A-11029) and Click-iT™ EdU Alexa Fluor™ 647 (C10340). Images were acquired on a Nikon upright microscope (60x).
Detection of alpha tubulin using Alexa Fluor 594 anti-mouse secondary antibody. Bovine pulmonary artery endothelial cells were labeled with Alexa Fluor™ 488 phalloidin (Cat. No. A12379) to stain F-actin and our mouse monoclonal anti–α-tubulin antibody (Cat. No. A11126) in combination with Alexa Fluor™ 594 dye–conjugated F(ab')2 fragment of goat anti–mouse IgG antibody (Cat. No. A-11020) to stain microtubules. The multiple-exposure image was acquired using bandpass filter sets appropriate for Texas Red™ dye and fluorescein.
- A-11001 was used in immunohistochemistry to study the role of autophagy in normothermic and hypothermic spinal cord ischemia. Fujita S, Sakurai M, Baba H et al. (2015) Autophagy-mediated stress response in motor neurons after hypothermic spinal cord ischemia in rabbits. J Vasc Surg 65(5):1312–1319.
- A-11029 was used in immunocytochemistry to assess the effects of fucoidan on diabetic nephropathy related to spontaneous diabetes. Wang Y, Nie M, Lu Y et al. (2015) Fucoidan exerts protective effects against diabetic nephropathy related to spontaneous diabetes through the NF-κB signaling pathway in vivo and in vitro. Int J Mol Med 35(4):1067–1073.
- A-21202 was used in immunohistochemistry–frozen sections to study the Pavlovian-instrumental transfer effect of the rostral medial ventral pallidum region innervated by the nucleus accumbens shell. Leung BK and Balleine BW (2015) Ventral pallidal projections to mediodorsal thalamus and ventral tegmental area play distinct roles in outcome-specific Pavlovian-instrumental transfer. J Neurosci 35(12):4953–4964.
How to find a secondary antibody
Step 1: Search for the target species of interest (e.g., “anti-mouse” or “goat anti-rabbit” or “alexa fluor 488”).
Step 2: Narrow results by host species, conjugate, application, and other criteria using side filters.
Thermo Scientific™ Superclonal™ secondary antibodies represent a breakthrough in recombinant antibody technology designed to provide precise and accurate detection of mouse, rabbit and goat primary antibodies in a variety of applications. Each Superclonal secondary antibody is formulated and optimized to help achieve excellent results in ELISA, western blot, and cell imaging.
- Manning, C. F., Bundros, A. M., & Trimmer, J. S. (2012) Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance. PLoS ONE, 7(6). doi:10.1371/journal.pone.0038313
- MGI – Mouse Vertebrate Homology. (2016) Retrieved June 30, 2016, from http://www.informatics.jax.org/homology.shtml
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