- Mary Johnson Ph. D.mary at labome dot comSynatom Research, Princeton, New Jersey, United States
This is a review about secondary antibodies used in peer-reviewed publications. Labome also reviews and summarizes primary antibodies cited in publications, with over 10,000 publications manually curated. The antibody users can search the reviews for primary antibodies against specific genes by using the search box above. A few sample reviews are listed here, beta actin, alpha tubulin, integrin antibody, phosphotyrosine antibody, and hemagglutinin antibody.
In order to help Labome.com visitors identify the most suited secondary antibodies, Labome conducted two surveys over the years. In the 2012 survey, information about 163 instances of secondary antibodies from 105 publications from 21 journals (mainly the journal Science) were collected and organized. Table 1 and 2 indicate the publication years and major journals of these 105 publications.
|Mol Cell Biol||6|
|Proc Natl Acad Sci U S A||2|
|J Cell Biol||2|
|Mol Biol Cell||2|
In an earlier 2008 survey, information about secondary antibodies from 99 recent publications in the journals Science and Journal of Biological Chemistry were collected and cataloged. The publications were randomly selected. Their authors came from more than 80 universities or research institutes in 16 countries. Data included host species, reactive species, Ig class, Ig subclass, conjugation, experimental methods, antibody dilution, and suppliers. Quite often, the relevant information were not available in these articles. In such cases, the corresponding authors were contacted and their clarifications if any were incorporated. For the list of the publications and detailed information about secondary antibodies used, please see here.
|mouse||13||            |
|rabbit||12||           |
Primary antibodies tend to be raised in goats, mice, rabbits, rats, and sheep, with mice and rabbits the most common ones. The reactive species of secondary antibodies correlate well with the host species of primary antibodies, with rabbits and mice as the dominant types.
|mouse||38||                                     |
|rabbit||30||                             |
|rat||5||    |
|goat||5||    |
Production of secondary antibodies requires species different from those of primary antibodies. Goats, donkeys, rabbits, sheep, and mice (for monoclonal secondary antibodies) are the hosts of choice for secondary antibodies. Among the 163 instances of secondary antibodies, 33 of them indicated the host species of the secondary antibodies. Table 3 lists the host species, indicating goat had been the predominant species for secondary antibodies. There were 79 instances where the reactive species were indicated, Table 4 lists the reactive species, indicating that, no surprise here, mouse and rabbit are the two major target species.
|IgG||9||        (IgG1),  (Fc gamma fragment)|
Anti-IgG secondary antibodies are most widely used. Among the 12 instances where an Ig class is indicated, 9 cases were IgG (Table 5). This is not surprising since most monoclonal and polyclonal primary antibodies are IgG class immunoglobulins. In addition, anti-IgG secondary antibodies can recognize various IgG subtypes. If the class and/or subclass of a primary antibody is unknown, an anti-IgG secondary antibody may be a good default choice.
Secondary antibodies specific to IgG subclasses (e.g. anti-IgG1, anti-IgG2, anti-IgG2a, anti-IgG2b) appear to be mainly used in double labeling experiments, including immunocytochemistry, immunohistochemistry, and flow cytometry.
Secondary antibodies tend to be against the whole IgG molecule. Those agasint F(ab')2 fragments or Fab fragment are used, for instances, when binding of secondary antibodies to Fc receptors on cell surfaces should be avoided, or there is endogenous immunoglobulin.
An article "Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance" by Dr. Trimmer's lab in University of California Davis tested the anti-mouse IgG subclass-specific secondary antibodies vs. general anti-mouse IgG seconday antibodies in immunoblots, immunohisto- and immunocyto-chemistry, and microplate binding assays and found that common anti-mouse heavy and light chain secondary antibodies "have detection bias away from the IgG1 mouse monoclonal subclass, which is 65-70% of available mouse monoclonals. This leads to lower detection levels of a majority of the monoclonal mouse antibodies used and decreased signal to noise ratios." The authors suggest that subsclass-specific anti-mouse secondary antibodies should instead be used to signficantly increase the detection of primary antibodies, and each lab should at least maintain the inventory of secondary antibodies against mouse IgG1, IgG2a and IgG2b subclasses . Dr. Trimmer leads the Neuromab group in UC Davis, and Labome visitors can search for Neuromab antibodies at Labome, using the search box above.
Secondary antibodies are used in all types of immunoassays, most often in Western blot, immunohistochemistry, and immunocytochemistry, and occasionally in immunoprecipitation, ELISA, and fluorescent in situ hybridization (Table 6). Basic research, clinical analysis, and disease diagnosis use secondary antiobodies in ELISA and flow cytometry assays.
|Alexa Fluor 488||2|
|Alexa Fluor 568||2|
|Alexa Fluor 594||1|
|Alexa Fluor 680||1|
|IR Dye 680||2|
|IR Dye 800||4|
Secondary antibodies serve two purposes, one is to amplify the signal, and another one is to enable easy detection. Without secondary antibodies, the primary antibodies would be conjugated. Most secondary antibodies in the survey were conjugated. The common conjugates of secondary antibodies were horseradish peroxidase, Alexa Fluor 488, and Cy3. Table 7 lists all the conjugates in the survey.
The choice of conjugates of secondary antibodies depends on specific application. For Western blot, the conjugate was likely to be horseradish peroxidase (HRP), alkaline phosphatase (AP), and fluorescent dyes like IRDye (e.g. IRDye 680, IRDye 800) and Alexa Fluor dye (e.g. Alexa 680 ) series (Table 8). HRP conjugated secondary antibodies from Amersham Biosciences, Bio-Rad, Cell Signaling, GE Healthcare Bio-Science, Jackson ImmunoResearch Laboratories, LI-COR Biotechnology, Pierce Biotechnology, Santa Cruz Biotechnology, Sigma, and Invitrogen(Zymed Laboratories) were often used in Western blot, and AP conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Sigma, Bio-Rad, and DAKO.
|Alexa Fluor 488||25|
|Alexa Fluor 546||1|
|Alexa Fluor 555||6|
|Alexa Fluor 568||5|
|Alexa Fluor 594||4|
|Alexa Fluor 647||5|
|Alexa Fluor 633||2|
For immunofluorescence (immunocytochemistry and immunohistochemistry), Alexa Fluor dye series of conjugates (e.g. Alexa Fluor 488, 546, 568, 594, 596, 647, 633), Cyanine dye series of conjugates (e.g. Cy2, Cy3, and Cy5), FITC, and rhodamine red are likely used (Table 9), with Invitrogen, Jackson Immunoresearch Laboratories, and Sigma as the main suppliers.
Secondary antibodies conjugated with FITC, PE, or Alexa Fluor 647 were used in flow cytometry; those conjugated with HRP, AP, and biotin were used in ELISA assays; and unconjugated secondary antibodies were used for protein isolation in immunoprecipitation.
|Invitrogen / Molecular Probes / Life Tech||64|
|Jackson Immuno Research||23|
|Santa Cruz Biotechnology||7|
|Sigma Chemical Co||5|
|Calbiochem / EMD Millipore||4|
|R & D Systems||1|
|Thermo Fisher Pierce||1|
Many high quality reagent providers supply secondary antibodies to both the research and diagnostics communities. The relatively small number of publications in this survey precludes an extensive sampling of suppliers. Invitrogen (including Molecular Probes, Biosource International, and Zymed Laboratories), Jackson ImmunoResearch Laboratories, Sigma, GE Healthcare Bio-Science (including Amersham Biosciences), Bio-Rad, Santa Cruz Biotechnology were the most cited suppliers (Table 10).
- Berarducci B, Rajamani J, Reichelt M, Sommer M, Zerboni L, Arvin A. Deletion of the first cysteine-rich region of the varicella-zoster virus glycoprotein E ectodomain abolishes the gE and gI interaction and differentially affects cell-cell spread and viral entry. J Virol. 2009;83:228-40 pubmed publisher
- Xu L, Chen Y, Song Q, Xu D, Wang Y, Ma D. PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and DNA damage-induced apoptosis. Neoplasia. 2009;11:345-54 pubmed
- Lomeli H, Starling C, Gridley T. Epiblast-specific Snai1 deletion results in embryonic lethality due to multiple vascular defects. BMC Res Notes. 2009;2:22 pubmed
- Zermeño V, Espindola S, Mendoza E, Hernandez-Echeagaray E. Differential expression of neurotrophins in postnatal C57BL/6 mice striatum. Int J Biol Sci. 2009;5:118-27 pubmed
- Bowes A, Khan M, Shi Y, Robertson L, Werstuck G. Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis. Am J Pathol. 2009;174:330-42 pubmed publisher
- IgA isotype control
- IgG1 isotype control
- IgG2a isotype control
- IgG2b isotype control
- IgG3 isotype control
- IgM isotype control
- anti-bovine secondary antibody
- anti-chicken secondary antibody
- anti-dog secondary antibody
- anti-goat secondary antibody
- anti-guinea pig secondary antibody
- anti-hamster secondary antibody
- anti-horse secondary antibody
- anti-mouse IgA secondary antibody
- anti-mouse IgD secondary antibody
- anti-mouse IgE secondary antibody
- anti-mouse IgG H+L secondary antibody
- anti-mouse IgG kappa secondary antibody
- anti-mouse IgG1 secondary antibody
- anti-mouse IgG2a secondary antibody
- anti-mouse IgG2b secondary antibody
- anti-mouse IgG2c secondary antibody
- anti-mouse IgG3 secondary antibody
- anti-mouse IgM secondary antibody
- anti-mouse kappa secondary antibody
- anti-mouse lambda secondary antibody
- anti-pig secondary antibody
- anti-rabbit secondary antibody
- anti-rat IgA secondary antibody
- anti-rat IgG H+L secondary antibody
- anti-rat IgG1 secondary antibody
- anti-rat IgG2a secondary antibody
- anti-rat IgG2b secondary antibody
- anti-rat IgG2c secondary antibody
- anti-rat IgM secondary antibody
- anti-sheep secondary antibody
- chicken isotype control
- goat isotype control
- hamster isotype control
- human isotype control
- isotyping kit
- rabbit isotype control
- sheep isotype control