Biotinylated secondary antibodies: detect low-abundance proteins
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Learn how to use biotinylated secondary antibodies within the avidin-biotin complex method, and labeled streptavidin-biotin method.
Biotin forms large complexes with avidin or streptavidin for signal amplification. Using either the avidin-biotin complex (ABC) method or labeled streptavidin-biotin (LSAB) method with biotinylated secondary antibodies can amplify signal in immunohistochemisty (IHC) or ELISA. Due to the conjugation of multiple biotin molecules to a secondary antibody, biotinylated secondary antibodies allow easy detection of proteins expressed at low levels.
Contents:
- What is biotin?
- Methods for using biotinylated antibodies
- Aiding signal amplification
- Which method should I use?
- Limitatios of biotinylated antibodies
- Find the right product for your research
What is biotin?
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 secondary antibodies
See Biotinylation Kit - Lightning-Link®
Methods for using biotinylated antibodies
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:
| Direct | Indirect | ABC | LSAB | |
| First | Conjugated primary antibody | Unlabeled primary antibody | Unlabeled primary antibody | Unlabeled primary antibody |
| Second | Conjugated secondary antibody | Biotinylated secondary antibody | Biotinylated secondary antibody | |
| Third | Avidin-biotin complex | Streptavidin complex |
Which 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:
| ABC | LSAB | Comments | |
| Specificity | Lower | Higher | Avidin 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. |
| Sensitivity | High | High | Both methods show greater sensitivity than direct or indirect detection. |
| Tissue penetration | Lower | Higher | The complex size in LSAB methods is smaller facilitating a greater tissue penetration. |
| Sample processing | More complex | Simpler | Both 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
Biotinylation kit: Biotin Conjugation Kit - Lightning-Link®
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.
