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For improved detection sensitivity, steptavidin-based amplification techniques are widely used in fluorescence imaging to detect biotinylated biomolecules such as primary and secondary antibodies, ligands and toxins, or DNA probes for in situ hybridization.

Streptavidin-based detection provides signal amplification for medium- and low-abundance targets with a simple workflow.

The family of biotin-binding proteins includes streptavidin, avidin and NeutrAvidin® protein, each protein binds four biotins per molecule with high affinity and selectivity. The most commonly used is streptavidin, which is non-glycosylated and exhibits low levels of nonspecific binding. Avidin is a highly cationic glycoprotein with an isoelectric point of about 10.5. Its positively charged residues and oligosaccharide component can interact nonspecifically causing background problems in some applications. NeutrAvidin® protein has been processed to remove the carbohydrate and lower its isoelectric point, which can result in reduced background staining.

When to use streptavidin-based amplification

Life Technologies offers the most complete range of detection technologies for fluorescence imaging. Using this suite of tools, you can select the optimal technique for your target abundance with complementary detection wavelengths that enable you to multiplex your experiment.

  • High abundance target—primary conjugate, no amplification needed
  • Medium abundance target—secondary conjugate, modest amplification
  • Medium-low abundance target—streptavidin conjugate, elevate the signal
  • Low-abundance target—enzyme amplification, complex process for maximum signal enhancement
 

Blocking endogenous biotin

Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes often produce substantial background signals when biotin–streptavidin or biotin–avidin detection systems are used to identify cellular targets. Endogenous biotin is particularly prevalent in mitochondria and in kidney, liver, and brain tissues.

The Endogenous Biotin-Blocking Kit helps to reduce background signals when biotin–streptavidin detection systems are used to identify targets in cells or tissue with high levels of endogenous biotin.

Figure 1. Alexa Fluor® 488 Streptavidin targeted to Golgi bodies also binds endogenous biotin (Left) resulting in non-specific labeling.  The Endogenous Biotin-Blocking Kit reduces non-specific labeling and green background signal (right).

How to select a streptavidin conjugate

Streptavidin is conjugated to a variety of fluorophores to image a range of target types. Traditional dye conjugates are utilized in most standard protocols, and allow you to image targets with a range of wavelengths. Alexa Fluor® conjugates provide bright signals and resistance to photobleaching with standard light cubes or wavelength settings. Qdot® conjugates provide the brightest signals for direct imaging with little or no photobleaching.

 

  Qdot® conjugates AlexaFluor® conjugates Standard dye conjugates
Optimum signal with standard light cubes or filters 
Resistance to photobleaching
Wide range of wavelengths
Ultimate brightness with optimized filters  
Cell surface and cytoplasmic targets
Nuclear targets 

Qdot® Conjugates for brightness and photostability

 Qdot® conjugates provide the brightest signals for direct imaging with little or no photobleaching, even in extended protocols. Standard light cubes or filters can be used to view Qdot® probes, but specialized filters are required to achieve optimal signal intensity. Qdot® conjugates provide intense signals for tracking cell surface and cytoplasmic targets; nuclear targets are more effectively imaged with organic labels such as Alexa Fluor® dyes.

Alexa Fluor ® conjugates for high-performance imaging

 AlexaFluor® streptavidin conjugates provide brightness and photostability using standard microscope settings, light cubes, or filters. The emission spectrum of Alexa Fluor® dyes covers the visible and IR range for easy multiplexing. Both nuclear and cytoplasmic structures are readily penetrated by these organic fluorophores for imaging and analysis.

Traditional dye conjugates for routine analysis

 Traditional dyes like fluorescein (FITC), tetramethylrhodamine (TRITC), and Texas Red® conjugated to a biotin-binding protein allow you to follow standard protocols and image both nuclear and cytoplasmic targets with a range of wavelengths. Traditional dye conjugates work best for relatively high-abundance targets where maximum brightness and photostability are not essential.

Imaging accessories

To help you get the optimal immunofluorescence imaging of fixed cells and tissue sections using our fluorescent streptavidin conjugates we offer Image-iT® FX Kits that provide all of reagents you need in one place.

Each kit provides sufficient materials to perform 50–100 assays and includes:

 

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