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The Avidin Biotin Complex Method
The VECTASTAIN ABC systems are extremely sensitive due to the form and number of active enzyme molecules associated with the preformed Avidin/Biotinylated enzyme Complex. This ABC complex takes advantage of two important properties of avidin: 1) an extraordinarily high affinity for biotin (over one million times higher than antibody for most antigens), and 2) four biotin-binding sites. These properties allow macromolecular complexes (ABCs) to be formed by mixing Avidin DH (Reagent A) with its paired biotinylated enzyme (Reagent B) prior to use. The ABC reagent, once formed, remains stable for many hours after formation and can be used for several days after preparation.
Biotinylated primary antibodies, secondaries, lectins, neuronal tracers, nucleic acids, and ligands can be effectively visualized in applications such as:
- Tissue staining
- Multiple labeling (Multiplex IHC)
- Western blotting
- Southern and northern blotting
- In situ hybridization detection (ISH)
- Enzyme immunoassays (ELISA)
- Neuronal tracing
All applications benefit from the high sensitivity, low background, reproducibility, and economy of the VECTASTAIN ABC system.
Choosing a VECTASTAIN ABC Kit
1) Choose an enzyme system:
The VECTASTAIN ABC Kits are available with a choice of two different enzyme systems
- Peroxidase (ABC, Elite® ABC, RTU or Kits)
- Alkaline Phosphatase (ABC-AP)
2) Choose the biotinylated secondary antibody:
To detect a biotinylated target, you will need only the ABC Reagent contained in the Standard Kit, followed by an appropriate substrate.
To detect an unlabeled primary antibody, you will need a biotinylated secondary antibody that binds to the primary antibody species chosen, the ABC Reagent, and an appropriate substrate. For example, to detect a primary antibody made in rabbit, the appropriate choice is a VECTASTAIN ABC Kit designated “Rabbit IgG”.
For additional versatility and convenience, Vector Laboratories offers secondary antibody products that recognize more than one species of primary antibody. Our Universal Biotinylated Anti-Mouse/Rabbit IgG secondary antibody (Cat. No. BA-1400) is a cocktail of biotinylated anti-mouse IgG and anti-rabbit IgG, designed for use with both rabbit and mouse primary antibodies. This antibody should not be used to stain tissues from rodents or rabbits due to its reactivity with IgG present in the tissues of these species. The Universal Pan-Specific Anti- Mouse/Rabbit/Goat secondary antibody (Cat. No. BA-1300) (which is included in our VECTASTAIN Universal Kits) is a cocktail of biotinylated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG that recognizes mouse, rabbit, and goat primary antibodies. This secondary antibody should not be used to stain rat, mouse, or other rodent, rabbit, goat, bovine, or sheep tissue due to potential reactivity with endogenous IgG.
3) Choose a convenient format:
Finally, several kits are available in prediluted, stabilized formats for convenience and ease of use. See kits designated R.T.U. or Ready-To-Use.
Consider Species Cross-Reactivity
When choosing the optimal detection system for your application, it is important to consider not only the species of the primary antibody but also the species of the tissue under examination. If the species of the primary antibody and the species of the tissue are closely related (e.g. rat and mouse), the biotinylated secondary antibody may cross react with endogenous IgG in the tissue section leading to background staining. The following three options minimize background staining in these instances:
- Biotinylate the primary antibody and detect with a Standard VECTASTAIN Elite ABC Kit, PK-6100 (only includes the avidin-biotin complex, or ABC reagent).
- Use a biotinylated secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely-related species (e.g. biotinylated anti-mouse IgG, rat adsorbed, Cat. No. BA-2001).
- Use the M.O.M.® Immunodetection System for applications of mouse primary antibodies on mouse tissue.
After choosing the VECTASTAIN ABC Kit for your application, select a substrate that matches the enzyme system of the kit.