A primary antibody's Fc domain is constant within the same animal species, therefore, a given secondary antibody can be used with any primary antibody of the same isotype and target species. This reduces the cost by labeling only one type of secondary antibody. In addition, secondary antibodies offer increased sensitivity through the signal amplification.
At GenScript, secondary antibodies are purified from the serum of several hosts, including goat, donkey, mouse, rabbit, chicken and sheep. Secondary antibodies are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions. The conjugated secondary antibodies can be used for various immunology analysis and protein assays.
GenScript also offers over 1000 highly specific monoclonal and polyclonal primary antibodies. Click here to look for primary antibodies for your protein of interest.
Step 1. The target species of the secondary antibody need to match the host of the primary antibody
Step 2. The immunogen of the secondary antibody need to match the class (isotype), subclass or the fragment of the primary antibody. Select an appropriate host species for the secondary antibody.
- Primary antibody: IL-8 Antibody, mAb, Mouse (Cat.no A00044),
- Host: mouse
- Ig Subclass: IgG1
- Secondary antibody: Goat Anti-Mouse IgG Antibody (H&L) [HRP], pAb (Cat.no A00160)
- Host: Goat
- Target species: mouse
- Immunogen: Full length mouse IgG (purified)
- It reacts with All classes, heavy and light chains (H&L) and fragments of mouse IgG.
Immunoglobulin (Ig) Types of Mammals
|Ig Class (Isotype)||Subclass||Heavy Chain||Light Chain||Complexes|
|Ig A||Ig A1, Ig A2||α1, α2||κ or λ||dimer|
|Ig G||Ig G1, Ig G2a, Ig G2b, Ig G3, Ig G4||γ1, γ2, γ3, γ4||monomer|
Step 3. Choose the label of the secondary antibody according to your application.
|Application||Label Type||Label Options|
Alkaline Phosphatase (AP),
Horseradish Peroxidase (HRP)
|Western Blot (WB)|
|Immunohistochemistry (IHC)||Enzyme/Fluorochrome||Biotinylated secondary antibodies – avidin/streptavidin conjugated to enzyme/fluorochrome|
Step 4. Consider other specificity issues of the secondary antibody
If working with tissues or cells that have Fc receptors (spleen, blood, hematopoietic cells, leukocytes, etc.), choose a F(ab) or F(ab')2 fragment to eliminate non-specific binding.
F(ab')2 fragment secondary antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact the hinge region. Two F(ab) fragments and an Fc fragment are generated if digested by papain.
Pre-adsorbed (also cross-adsorbed)
The pre-adsorption process reduces the risk of cross reactivity between the secondary antibody and endogenous immunoglobulins present on cell and tissue samples. The process involves passing the secondary antibody over immobilized serum proteins from potentiAlly cross reactive species.
Example: Goat Anti-Mouse IgG (H&L) [TRITC], pAb (Cat.no A10071) is pre-adsorbed and has Min X Bv Ch Gt GP Ham Hs Hu Rb Rt & Sh Serum Proteins.
Polyclonal antibodies are sufficient for most needs. Monoclonal antibodies can minimize background, cross reactivity and improve the lot-to-lot consistency.
Mouse Anti-Rabbit IgG Antibody (M205) [HRP], mAb (Cat.no A01827-200) only recognizes native rabbit primary IgG antibodies and does not react with denatured or reduced rabbit IgG heavy or light chain. Immunoprecipitation and Western blots often suffer from primary antibody heavy and light chain contamination at approximately 50 and 25 kDa, which is caused by poor specificity of conventional polyclonal secondary antibodies derived from goat or other species.
- Is Catalog No. A00166 (Goat Anti-Human IgG Antibody (H&L) [HRP], pAb) specific for human IgG? Does it cross-react with non-human primate or rodents?
Catalog No. A00166 (Goat Anti-Human IgG Antibody (H&L) [HRP], pAb) recognizes human IgG, but it has not yet been tested for cross-reactivity with other species.
- Does Catalog No. A00160 (Goat Anti-Mouse IgG Antibody (H&L) [HRP], pAb) cross react with rabbit?
Catalog. No A00160 (Goat Anti-Mouse IgG Antibody (H&L) [HRP], pAb) has not yet been tested for cross-reactivity with rabbit.