All tags Secondary antibodies Detect low-abundance proteins with biotinylated antibodies

Detect low-abundance proteins with biotinylated antibodies

Biotin forms large complexes with avidin or streptavidin for signal amplification. Learn about the different methods that use biotin-conjugated antibodies.

Biotin is a naturally occurring vitamin with important roles in numerous biological processes. There is a strong affinity between biotin and avidin or streptavidin. Avidin is a glycoprotein found in some tissues of birds, reptiles and amphibians. Streptavidin is isolated from Streptomyces avidinii.  Both proteins can easily form large complexes by binding up to four biotins per molecule.  Thus, conjugation of biotin to antibodies and reporter enzymes or fluorophores provide a powerful means to amplify signals.  These characteristics have made methods based on biotinylated antibodies ideal for the detection of low-abundance proteins.

See biotinylated secondaries

Biotinylated antibodies are used in two methods:

  • Avidin-biotin complex (ABC) method: large avidin-biotin complexes linked through reporter enzymes are incubated with biotinylated antibodies.  The signal is amplified due to the high enzyme-to-antibody ratio.
  • Labeled streptavidin-biotin (LSAB) method: the signal is amplified through incubation of the biotinylated antibody with complexes made of streptavidin and reporter enzymes. Complexes in the LSAB method are smaller than in the ABC method.

An additional layer for extra signal amplification

Unlike other methods (see direct vs indirect methods), these techniques add another layer for signal amplification. The additional layer associates a primary antibody with multiple enzymes or fluorophores, increasing considerably the sensitivity. For instance, if using biotinylated secondary antibodies:


DirectIndirectABCLSAB
FirstConjugated primary antibodyUnlabeled primary antibodyUnlabeled primary antibodyUnlabeled primary antibody
Second
Conjugated secondary antibodyBiotinylated secondary antibodyBiotinylated secondary antibody
Third

Avidin-biotin complexStreptavidin complex

What method should I use?

LSAB methods have become more popular than ABC over the last years. The main reason is the lower non-specific binding of streptavidin proteins. But there are additional benefits to LSAB methods as highlighted below:


ABCLSABComments
SpecificityLowerHigherAvidin may show non-specific binding due to its carbohydrate moieties and its high isoelectric point (pI). In contrast, streptavidin lacks carbohydrate moieties and has a more neutral pI.
SensitivityHighHighBoth methods show greater sensitivity than direct or indirect detection.
Tissue penetrationLowerHigherThe complex size in LSAB methods is smaller facilitating a greater tissue penetration.
Sample processingMore complexSimplerBoth methods require three incubations steps, but ABC methods require an additional incubation of avidin with the reporter enzyme.

Limitations of using biotinylated antibodies

Despite their wide adoption, these methods also come with their limitations. The presence of endogenous biotin in tissues can significantly increase background signal. Therefore, blocking endogenous biotin is particularly important in tissues with high expression of the molecule such as the liver and kidney. Note that non-biotin-based detection methods are recommended with frozen tissue sections due to their high amounts of endogenous biotin.

Find the right product for your research

We provide both the biotinylated antibodies and the biotin-binding proteins you need. Our range includes:

Primary antibodies:           Rabbit polyclonals           Mouse monoclonals

Secondary antibodies:     Anti-rabbit antibodies      Anti-mouse antibodies

Proteins:                              Avidins                               Streptavidins

The images below show examples of the use of biotinylated antibodies in immunohistochemistry (IHC) and ELISA.

IHC image of Anti-Histone H4 [EPR16599] – ChIP Grade RabMAb antibody (ab177840) staining in a section of FFPE human colon tissue. Goat Anti-Rabbit IgG H&L (Biotin) (ab207995) was used as the secondary antibody. Endogenous biotin was blocked using Endogenous Avidin/Biotin Blocking Kit (ab64212). Detection was via an HRP conjugated ABC system and DAB was used as the chromogen. The section was then counterstained with haematoxylin. The inset negative control image is taken from an identical assay without primary antibody. For further details, visit the datasheet here.

ELISA testing of Goat Anti-Rabbit IgG H&L (Biotin) (ab207995) using wells coated with serially diluted mouse IgG. Antibody binding was detected with Streptavidin-HRP (ab7403). Signal was developed by TMB substrate. For further details, visit the datasheet here.


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