What is a secondary antibody?
A secondary antibody is an antibody that can bind to a primary antibody. It is mainly used to detect the presence of the primary antibody. The primary antibody specifically binds to the antigen, and the secondary antibody specifically binds to the primary antibody. In other words, the Fc region or Fab fragment of the primary antibody can act as an antigen to stimulate the body to produce antibodies. That is to say, the primary antibody is mainly IgG class immunoglobulins, so the secondary antibody is an anti-IgG antibody.
The role of secondary antibodies
The primary antibody specifically binds to the target protein, and the primary antibody needs to be labeled with a luminescent substance or a chromogenic group. It is not possible to see with the naked eye whether the primary antibody bind the protein. However, the primary antibody is relatively specific, and each one is labeled at a high cost. It is common to use a labeled secondary antibody that can bind to the primary antibody and has a detectable label (such as a fluorescent, radioactive, chemiluminescent, or chromogenic group). So the current design generally has the secondary antibody with a detectable label to detect the primary antibody, while the primary antibody recognizes the substrate. In this way, when the primary antibody binds to the substrate, it can be detected through the secondary antibody.
For example, the ECL luminescence principle: In the ECL substrate, there are H2O2 and luminol (and its derivatives), which emit fluorescence under the action of HRP (horseradish peroxidase).
Key points for using secondary antibodies
The secondary antibody is an antibody produced by immunizing mammals (usually immunizing goats, horses, donkeys, mules, and guinea pigs, etc.) with the immunoglobulin IgG of a certain subtype of the primary antibody species (such as rabbits, mice), so the secondary antibody only binds to a specific species and subtype of the primary antibody. For example, sheep anti-mouse (secondary antibody) will specifically bind to mouse monoclonal (primary antibody) and will not bind to rabbit polyclonal or rat monoclonal.
Coupling marks of secondary antibodies
Different experimental methods will result in different coupling groups for secondary antibodies, which are mainly enzymes (horseradish peroxidase/HRP and alkaline phosphatase/AP), fluorescent groups (FITC, Rhodamine, PE, Dylight, Alexa Fluor, cy3, etc.), biotin, gold particles, etc. Generally, HRP-labeled secondary antibodies are recommended for experiments such as WB, ELISA, IHC, and biotin-labeled secondary antibodies are recommended for IHC, while fluorescent labels such as Alexa Fluor 488, FITC, PE are used for IF, FC, etc.
How to choose the right secondary antibody
a. Immunoblotting (WB)
Select a secondary antibody labeled with horseradish peroxidase or alkaline phosphatase for traditional Western blotting;choose a secondary antibody labeled with the corresponding fluorophore for fluorescent Western blotting.
b.Immunohistochemistry (IHC/ICC)
B.For ICC (Immunocytochemistry), select a secondary antibody labeled with Biotin.
C. Immunofluorescence (IHF/ICF)
Immunoprecipitation often allows the choice of using a secondary antibody that is either Fc or Fab specific. Such secondary antibodies can avoid interference from the heavy chain (molecular weight 55 kDa) or the light chain (molecular weight 25 kDa).
Recommended Product Information
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Label Group |
Product Number |
Product Name |
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Unmarked |
34850ES |
Goat Anti-Rabbit lgG(H+L) |
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34851ES |
Goat Anti-Mosue lgG(H+L) |
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34847ES |
Donkey Anti-Human IgG(H+L) |
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HRP |
33101ES |
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) |
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33201ES |
Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) |
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33301ES |
Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) |
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33401ES |
Peroxidase AffiniPure Goat Anti-Guinea Pig IgG(H+L) |
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33501ES |
Horseradish Peroxidase Goat Anti-Human IgG (H+L) |
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33701ES |
Peroxidase AffiniPure Rabbit Anti-Goat IgG (H+L) |
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33801ES |
Peroxidase AffiniPure Rabbit Anti-Sheep IgG (H+L) |
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34201ES |
Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) |
|
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34101ES |
Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L) |
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34301ES |
Peroxidase AffiniPure Donkey Anti-Goat IgG (H+L) |
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33101ES |
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) |
|
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Green Fluorescence |
33106ES |
YSFluor™ 488 Goat Anti-Rabbit IgG (H+L) |
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33126ES |
YSFluor™ 488 Goat Anti-Human IgG (H+L) |
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33206ES |
YSFluor™488 Goat Anti-Mouse IgG (H+L) |
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33306ES |
YSFluor™ 488 Goat Anti-Bovine IgG (H+L) |
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33406ES |
YSFluor™ 488 Goat Anti-Guinea Pig IgG (H+L) |
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33706ES |
YSFluor™488 Rabbit Anti-Goat IgG (H+L) |
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33906ES |
YSFluor™ 488 Rabbit Anti-Mouse IgG (H+L) |
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34906ES |
YSFluor™488 Donkey Anti-Sheep IgG (H+L) |
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34106ES |
YSFluor™488 Donkey Anti-Mouse IgG (H+L) |
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34206ES |
YSFluor™488 Donkey Anti-Rabbit IgG (H+L) |
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34306ES |
YSFluor™ 488 Donkey Anti-Goat IgG (H+L) |
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34406ES |
YSFluor™ 488 Donkey Anti-Rat IgG (H+L) |
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34506ES |
YSFluor™488 Donkey Anti-Guinea Pig IgG (H+L) |
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34606ES |
YSFluor™488 Donkey Anti-Chicken IgG (H+L) |
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34706ES |
YSFluor™488 Rabbit Anti-Chicken IgY(H+L) |
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34806ES |
YSFluor™488 Donkey Anti-Human IgG (H+L) |
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34856ES |
FITC AffiniPure Goat Anti-Human IgG(H+L) |
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33107ES |
FITC-AffiniPure Goat Anti-Rabbit IgG (H+L) |
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33207ES |
FITC-AffiniPure Goat Anti-Mouse IgG (H+L) |
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33307ES |
FITC-AffiniPure Goat Anti-Rat IgG (H+L) |
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33707ES |
FITC-AffiniPure Rabbit Anti-Goat IgG (H+L) |
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33807ES |
FITC-AffiniPure Rabbit Anti-Sheep IgG (H+L) |
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Rhodamine |
33109ES |
Rhodamine (TRITC) Goat Anti-Rabbit IgG (H+L) |
|
33209ES |
Rhodamine (TRITC) Goat Anti-Mouse IgG (H+L) |
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33709ES |
Rhodamine(TRITC) AffiniPure Rabbit Anti-Goat IgG (H+L) |
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|
33210ES |
Rhodamine Red-X (RRX) Goat Anti-Mouse IgG (H+L) |
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Orange Fluorescence |
33112ES |
YSFluor™594 AffiniPure Goat Anti-Rabbit IgG (H+L) |
|
33212ES |
YSFluor™ 594 Goat Anti-Mouse IgG (H+L) |
|
|
33222ES |
YSFluor™ 594 Goat Anti-Human IgG (H+L) |
|
|
33412ES |
YSFluor™594 Goat Anti-Guinea Pig IgG (H+L) |
|
|
33312ES |
YSFluor™ 594 Goat Anti-Bovine IgG (H+L) |
|
|
33712ES |
YSFluor™594 Rabbit Anti-Goat IgG (H+L) |
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|
33812ES |
YSFluor™ 594 Rabbit Anti-Sheep IgG (H+L) |
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33912ES |
YSFluor™594 Rabbit Anti-Mouse IgG (H+L) |
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|
34712ES |
YSFluor™594 Rabbit Anti-Chicken IgY (H+L) |
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34112ES |
YSFluor™ 594 Donkey Anti-Mouse IgG (H+L) |
|
|
34212ES |
YSFluor™594 Donkey Anti-Rabbit IgG (H+L) |
|
|
34312ES |
YSFluor™ 594 Donkey Anti-Goat IgG (H+L) |
|
|
34412ES |
YSFluor™ 594 Donkey Anti-Rat IgG (H+L) |
|
|
34512ES |
YSFluor™594 Donkey Anti-Guinea Pig IgG (H+L) |
|
|
34612ES |
YSFluor™594 Donkey Anti-Chicken IgG (H+L) |
|
|
34812ES |
YSFluor™594 Donkey Anti-Human IgG (H+L) |
|
|
34912ES |
YSFluor™594 Donkey Anti-Sheep IgG (H+L) |
|
|
33208ES |
Cy3-AffiniPure Goat Anti-Mouse IgG (H+L) |
|
|
33308ES |
Cy3-AffiniPure Goat Anti-Rat IgG (H+L) |
|
|
33708ES |
Cy3-AffiniPure Rabbit Anti-Goat IgG (H+L) |
|
|
33808ES |
Cy3-AffiniPure Rabbit Anti-Sheep IgG (H+L) |
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|
34855ES |
Cy3-AffiniPure Donkey Anti-Goat IgG(H+L) |
|
|
Red Fluorescence |
33113ES |
YSFluor™647 Goat Anti-Rabbit IgG (H+L) |
|
33213ES |
YSFluor™ 647 Goat Anti-Mouse IgG (H+L) |
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|
33223ES |
YSFluor™647 Goat Anti-Human IgG (H+L) |
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33313ES |
YSFluor™ 647 Goat Anti-Bovine IgG (H+L) |
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|
33413ES |
YSFluor™ 647 Goat Anti-Guinea Pig IgG (H+L) |
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33713ES |
YSFluor™ 647 Rabbit Anti-Goat IgG (H+L) |
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33813ES |
YSFluor™647 Rabbit Anti-Sheep IgG (H+L) |
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|
33913ES |
YSFluor™ 647 Rabbit Anti-Mouse IgG (H+L) |
|
|
34713ES |
YSFluor™647 Rabbit Anti-Chicken IgY (H+L) |
|
|
34113ES |
YSFluor™ 647 Donkey Anti-Mouse IgG (H+L) |
|
|
34213ES |
YSFluor™ 647 Donkey Anti-Rabbit IgG (H+L) |
|
|
34413ES |
YSFluor™ 647 Donkey Anti-Rat IgG (H+L) |
|
|
34813ES |
YSFluor™647 Donkey Anti-Human IgG (H+L) |
|
|
34913ES |
YSFluor™647 Donkey Anti-Sheep IgG (H+L) |
References
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[6] Liu C, He X, Liu W, et al. Bi-allelic Mutations in TTC29 Cause Male Subfertility with Asthenoteratospermia in Humans and Mice[J]. The American Journal of Human Genetics, 2019, 105(6): 1168-1181. 34106ES60 IF:9.924
[7]Xu T, Ding W, Ao X, et al. ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening[J]. Redox biology, 2019, 20: 414-426.IF（7.126）（Alexa Fluor 488）
[8] Tan T, Wang Y, Wang J, et al. Targeting peptide-decorated biomimetic lipoproteins improve deep penetration and cancer cells accessibility in solid tumor. Acta Pharm Sin B. 2020;10(3):529-545. Alexa Fluor 488 labeled IgG (H+L)IF: 7.097
[9] A Photoclick Hydrogel for Enhanced Single〤ell Immunoblotting[J]. Advanced Functional Materials, 2020, 30.33112ES60 IF: 16.836
[10] Bingcheng Yi a,b,1, Lei Yuc,1, Han Tanga,et al. Lysine-doped polydopamine coating enhances antithrombogenicity and endothelialization of an electrospun aligned fifibrous vascular graft.. Applied Materials Today,2021. 33207ES60 IF:10.041