How to Choose the Right Secondary Antibody

How to Choose the Right Secondary Antibody

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)

A.For IHC (Immunohistochemistry), select a secondary antibody labeled with HRP or DAB;
B.For ICC (Immunocytochemistry), select a secondary antibody labeled with Biotin.
C. Immunofluorescence (IHF/ICF)
C. For IHF (Immunofluorescence on tissue sections), select a secondary antibody labeled with FITC; D. For ICF (Immunofluorescence on cells), select a secondary antibody labeled with FITC.
D. Immunoprecipitation and Co-Immunoprecipitation

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

Label Group

Product Number

Product Name

Unmarked

34850ES

Goat Anti-Rabbit lgG(H+L)

34851ES

Goat Anti-Mosue lgG(H+L)

34847ES

Donkey Anti-Human IgG(H+L)

HRP

33101ES

Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)

33201ES

Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L)

33301ES

Peroxidase AffiniPure Goat Anti-Rat IgG (H+L)

33401ES

Peroxidase AffiniPure Goat Anti-Guinea Pig IgG(H+L)

33501ES

Horseradish Peroxidase Goat Anti-Human IgG (H+L)

33701ES

Peroxidase AffiniPure Rabbit Anti-Goat IgG (H+L)

33801ES

Peroxidase AffiniPure Rabbit Anti-Sheep IgG (H+L)

34201ES

Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L)

34101ES

Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L)

34301ES

Peroxidase AffiniPure Donkey Anti-Goat IgG (H+L)

33101ES

Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)

Green Fluorescence

33106ES

YSFluorâ„¢ 488 Goat Anti-Rabbit IgG (H+L)

33126ES

YSFluorâ„¢ 488 Goat Anti-Human IgG (H+L)

33206ES

YSFluorâ„¢488 Goat Anti-Mouse IgG (H+L)

33306ES

YSFluorâ„¢ 488 Goat Anti-Bovine IgG (H+L)

33406ES

YSFluorâ„¢ 488 Goat Anti-Guinea Pig IgG (H+L)

33706ES

YSFluorâ„¢488 Rabbit Anti-Goat IgG (H+L)

33906ES

YSFluorâ„¢ 488 Rabbit Anti-Mouse IgG (H+L)

34906ES

YSFluorâ„¢488 Donkey Anti-Sheep IgG (H+L)

34106ES

YSFluorâ„¢488 Donkey Anti-Mouse IgG (H+L)

34206ES

YSFluorâ„¢488 Donkey Anti-Rabbit IgG (H+L)

34306ES

YSFluorâ„¢ 488 Donkey Anti-Goat IgG (H+L)

34406ES

YSFluorâ„¢ 488 Donkey Anti-Rat IgG (H+L)

34506ES

YSFluor™488 Donkey  Anti-Guinea Pig IgG (H+L)

34606ES

YSFluor™488 Donkey  Anti-Chicken IgG (H+L)

34706ES

YSFluorâ„¢488 Rabbit Anti-Chicken IgY(H+L)

34806ES

YSFluorâ„¢488 Donkey Anti-Human IgG (H+L)

34856ES

FITC AffiniPure Goat Anti-Human IgG(H+L)

33107ES

FITC-AffiniPure Goat Anti-Rabbit IgG (H+L)

33207ES

FITC-AffiniPure Goat Anti-Mouse IgG (H+L)

33307ES

FITC-AffiniPure Goat Anti-Rat IgG (H+L)

33707ES

FITC-AffiniPure Rabbit Anti-Goat IgG (H+L)

33807ES

FITC-AffiniPure Rabbit Anti-Sheep IgG (H+L)

Rhodamine

33109ES

Rhodamine (TRITC) Goat Anti-Rabbit IgG (H+L)

33209ES

Rhodamine (TRITC) Goat Anti-Mouse IgG (H+L)

33709ES

Rhodamine(TRITC) AffiniPure Rabbit Anti-Goat IgG (H+L)

33210ES

Rhodamine Red-X (RRX) Goat Anti-Mouse IgG (H+L)

Orange Fluorescence

33112ES

YSFluorâ„¢594 AffiniPure Goat Anti-Rabbit IgG (H+L)

33212ES

YSFluorâ„¢ 594 Goat Anti-Mouse IgG (H+L)

33222ES

YSFluorâ„¢ 594 Goat Anti-Human IgG (H+L)

33412ES

YSFluorâ„¢594 Goat Anti-Guinea Pig IgG (H+L)

33312ES

YSFluorâ„¢ 594 Goat Anti-Bovine IgG (H+L)

33712ES

YSFluorâ„¢594 Rabbit Anti-Goat IgG (H+L)

33812ES

YSFluorâ„¢ 594 Rabbit Anti-Sheep IgG (H+L)

33912ES

YSFluorâ„¢594 Rabbit Anti-Mouse IgG (H+L)

34712ES

YSFluorâ„¢594 Rabbit Anti-Chicken IgY (H+L)

34112ES

YSFluorâ„¢ 594 Donkey Anti-Mouse IgG (H+L)

34212ES

YSFluorâ„¢594 Donkey Anti-Rabbit IgG (H+L)

34312ES

YSFluorâ„¢ 594 Donkey Anti-Goat IgG (H+L)

34412ES

YSFluorâ„¢ 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

YSFluorâ„¢594 Donkey Anti-Human IgG (H+L)

34912ES

YSFluorâ„¢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)

34855ES

Cy3-AffiniPure Donkey Anti-Goat IgG(H+L)

Red Fluorescence

33113ES

YSFluorâ„¢647 Goat Anti-Rabbit IgG (H+L)

33213ES

YSFluorâ„¢ 647 Goat Anti-Mouse IgG (H+L)

33223ES

YSFluorâ„¢647 Goat Anti-Human IgG (H+L)

33313ES

YSFluorâ„¢ 647 Goat Anti-Bovine IgG (H+L)

33413ES

YSFluorâ„¢ 647 Goat Anti-Guinea Pig IgG (H+L)

33713ES

YSFluorâ„¢ 647 Rabbit Anti-Goat IgG (H+L)

33813ES

YSFluorâ„¢647 Rabbit Anti-Sheep IgG (H+L)

33913ES

YSFluorâ„¢ 647 Rabbit Anti-Mouse IgG (H+L)

34713ES

YSFluorâ„¢647 Rabbit Anti-Chicken IgY (H+L)

34113ES

YSFluorâ„¢ 647 Donkey Anti-Mouse IgG (H+L)

34213ES

YSFluorâ„¢ 647 Donkey Anti-Rabbit IgG (H+L)

34413ES

YSFluorâ„¢ 647 Donkey Anti-Rat IgG (H+L)

34813ES

YSFluorâ„¢647 Donkey Anti-Human IgG (H+L)

34913ES

YSFluorâ„¢647 Donkey Anti-Sheep IgG (H+L)

 

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