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Avidin and Streptavidin


 

 

Avidin and Streptavidin Overview

 

Rockland offers avidin and streptavidin  conjugated to enzymes and fluorescent dyes for use in combination with biotinylated secondary antibodies.  Avidin or Streptavidin and biotin systems are often deployed in research immunoassays like ELISA, western blotting, immunohistochemistry to amplify signal.  Both avidin and streptavidin are tetrameric proteins capable of binding 4 biotin groups to each molecule of avidin or streptavidin.  Below is an example of biotin-streptavidin signal enhancement for primary antibody detection in immunohistochemistry.

 

 

Enhanced Streptavidin

 

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Biotinylated Secondary Antibodies

 


 

Unique characteristics making Avidin or Streptavidin with Biotin ideal for diverse applications:


• High affinity of avidin or streptavidin for biotin (1015 L• mol-1).
• High specificity ensure binding is only to target of interest.
• Tetravalent binding increase detection sensitivity.
• Small size of biotin does not affect biological activity, i.e. enzymatic catalysis or antibody binding.
• Exceptional stability of avidin and streptavidin are ideal to be chemically modified.

Applications


For western blotting use Streptavidin Peroxidase Conjugate S000-03
For immunofluorescence microscopy use Streptavidin Fluorescein Conjugate S000-02
For chemiluminescent western blotting use Avidin Peroxidase Conjugate A003-03

 


 

Classes of compounds used to incorporate biotin:


To be functional the avidin or streptavidin and biotin systems require one component to be biotinylated.  The following types of molecules can be derivatized to include a biotin moiety:
• Proteins, i.e. antibodies, analytes, enzymes
• Carbohydrates
• Nucleic Acids
• Small molecule analytes, i.e. therapeutics, toxins, drugs of abuse



Basic Configurations of the Avidin or Streptavidin and Biotin Detection System:


The avidin-biotin system can be used to detect a diverse group of targets.  The interaction of avidin or streptavidin with biotin can be configured in multiple configurations that offer assay flexibility and increased sensitivity.  The diagram below illustrates the most common configurations used in basic immunoassays.

 

Solid Support

 

The target (T) is bound to a solid phase, i.e. ELISA plates, agarose beads, tissue sections.  The binder (Ab) in this example is an antibody, but can be any of the classes of compounds listed above.  The binder is biotinylated (B) randomly at multiple sites.  Often 10-15 biotin moiety are coupled to an IgG secondary antibody.  In (1) streptavidin (S) is coupled to a label (L) i.e. an enzyme, fluorochrome, nucleic acid, or analyte, non-covalently binds to one or more biotin moieties on the binder (Ab).   In (2) unconjugated streptavidin (S) is used to link multiple labels (L) that are also derivatized with biotin (B). 
While these examples are only the most basic configurations and are over simplified for clarity, they illustrate the diversity of the biotin-avidin system used in many immunoassays supported by Rockland.


 

Uses of Avidin/Streptavidin reagents:


Immunofluorescent microscopy
• Coupling to colloidal gold
Flow cytometry
• Fluorescent in situ hybridization (FISH)
• Anti-nuclear antibody (ANA) assays
• High Throughput Screening (HTS)

Avidin Streptavidin Biotin

 

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