Biotin and Streptavidin

The biotin-streptavidin complex is one of the most popular tagging systems for the conjugation of biomolecules, such as proteins, lipids, and nucleic acids, as well as that of synthetic molecules, such as fluorescent labels. It has found strong success in the area of sample preparation as a core part of many purification systems and plays a critical role in many detection systems for instruments such as microscopy and flow cytometry.

Biotin-Streptavidin Conjugation System

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The widespread adoption of the biotin-streptavidin conjugation system is primarily due to two factors. The first is the relatively small size of the biotin and streptavidin molecules themselves, which allows for extensive binding to biologically active macromolecules, such as antibodies, without impedance to their functions (i.e., an antibody's binding site). The second is the specificity with which biotin and streptavidin bind to each other and the strength of the subsequent bond, which allows for powerful, stream-lined bioassay applications. Streptavidin tetramers have an extraordinarily high binding affinity for biotin with a dissociation constant (K_d) of approximately ~10^-14 mol/L. This tight and specific binding is rapid and can withstand extremes in pH, temperature, organic solvents, and denaturing reagents.

Advantages of the biotin-streptavidin system

A key benefit of the biotin-streptavidin system is its ability to improve detection sensitivity. This, in large part, is due to the tetrameric conformation of streptavidin. One streptavidin protein can bind four biotin molecules with high affinity and selectivity. This multiplicity enables the amplification of weak signals and improves the detection sensitivity for medium- and low-abundance targets in mammalian cells or tissues with a simple workflow.

Another key benefit is the versatility of the biotin-streptavidin system. Because streptavidin can be conjugated to various reporter tags, it can be readily incorporated into virtually every immunoassay. For example, enzyme conjugates of streptavidin are widely used in enzyme-linked immunosorbent assays (ELISAs), while fluorescently labeled streptavidin, such as iFluor® 488 streptavidin, are widely used in cell surface labeling, fluorescence-activated cell sorting (FACS) and other fluorescence imaging applications.

Applications

• Enzyme-linked immunosorbent assay (ELISA)
• Immunohistochemistry (IHC)
• Power Styramide™ Signal Amplification, a superior replacement for tyramide
• Immunoblotting
• Immunofluorescence microscopy
• Cell surface labeling
• Affinity purification
• Fluorescence-activated cell sorting (FACS)
• Flow cytometry

Biotin

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Biotin is a small 244-dalton hapten molecule. Its high binding affinity for streptavidin is commonly exploited to detect and monitor biological targets of interest. Biotin exhibits two characteristics that make it ideal for bioconjugate development. First, biotin is relatively small in size. This permits labeling multiple biotin tags to a single protein or antibody without significantly impeding its biological reactivity. The second is the valeric acid side chain of the biotin molecule. This chain can be derivatized to facilitate the incorporation of various reactive groups used to conjugate biotin to proteins without altering biotin's binding affinity for streptavidin.

AAT Bioquest offers biotinylated secondary antibodies, proteins, nucleotides, and other small molecules for use in streptavidin-based amplification techniques. For assays in which a biotinylated probe is unavailable, we provide many biotinylation reagents and kits that enable researchers to chemically label proteins, nucleic acids, and surface materials to make custom biotinylated reagents.

Featured Products

• Fluorescent biotin derivatives
• Biotinylated secondaries
• Biotinylated nucleotides
• Biotinylated amino acids
• Reactive-biotin derivatives
• ReadiLink™ Protein Biotinylation Kit
• Amplite® Colorimetric Biotin Quantitation Kit

Fluorescent Biotin Derivatives

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Fluorescent biotin derivatives contain a fluorophore and a biotin moiety in the same molecule. These reagents are used for detecting and quantifying biotin-binding proteins by fluorescence. The strong quenching associated with streptavidin binding to fluorescent biotin can be used to precisely measure the concentration of streptavidin (or avidin).

1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).

Biotinylated Secondary Antibodies

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We offer biotinylated secondary antibodies to use with streptavidin-based amplification techniques. A key advantage to using a biotinylated secondary is the flexibility to use the same secondary antibody across multiple applications by using a different streptavidin conjugate.

Readily available streptavidin conjugates

• HRP-streptavidin (imaging requires an HRP substrate)
• AP-streptavidin (imaging requires an AP substrate)
• iFluor®-streptavidin
• mFluor™-streptavidin
• APC, PE & tandem dye-streptavidins (The intense bright fluorescence and rapid photobleaching of phycobiliproteins are excellent for flow cytometry applications)

Biotinylated Nucleotides

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Biotin-modified nucleoside triphosphate analogs, such as dUTP and dCTP, can be enzymatically incorporated into DNA or RNA fragments for use in fluorescence in situ hybridization (FISH), DNA arrays, microarrays, and other hybridization techniques. Standard enzymatic non-radioactive DNA labeling reactions include 3''-end labeling, cDNA labeling, nick translation, PCR, and random prime labeling. Each biotinylated nucleotide contains either an 11-, 14-, 16- or 20-atom spacer between the biotin and its attachment point on the nucleotide. This facilitates its detection and signal amplification by fluorophore and enzyme streptavidin conjugates or agarose and magnetic beads.

Biotinylated Amino Acids

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Biotinylated peptides are suitable for immunodetection studies and biomedical screening assays that require immobilization onto streptavidin or avidin coated microtitre plates, beads, or membranes. Biotin can be linked to the N-terminus of peptides or the side chains of lysine (Lys), glutamic acid (Glu), or aspartic acid (Asp) during Fmoc solid phase synthesis. Incorporating a PEG-spacer in both FMOC-Glu(biotinyl-PEG)-OH and FMOC-Asp(biotinyl-PEG)-OH reduces steric hindrance between target peptide and streptavidin, leading to better biotin binding. The hydrophilic nature of the PEG-spacer minimizes non-specific interactions and improves the solubility of the reagent and biotinylated peptide.

Reactive-Biotin Derivatives

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Reactive biotin derivatives contain reactive moieties, such as succinimidyl esters or maleimides, that facilitate labeling biotin onto proteins, antibodies, and other macromolecules. When selecting a reactive biotin derivative, consider the following:

• Funtional group targeting - specific reactive moieties target and bind to their respective functional groups for targeted or non-selective biotinylation of proteins and other macromolecules (e.g., succinimidyl esters target primary amines and maleimides target thiol or sulfhydryl groups).
• Solubility - this influences biotinylation of the target protein or macromolecule.
• Spacer Arm Length (PEG) - enhance detection sensitivity of the target protein and reduces steric hindrance.

ReadiView™ Biotinylation Reagents

Determining a protein's degree of biotinylation is quite challenging because the intrinsic absorption of biotin is difficult to discern from proteins and nucleic acids. To facilitate this determination, we offer a series of chromophoric reactive biotin derivatives, ReadiView™ biotin.

These novel biotinylation reagents contain a specially designed 'Color Tag (CT)' optimally positioned between two spacer arms. These spacer arms reduce steric hindrance and improve water solubility, while the CT tag makes the degree of biotinylation readily quantifiable by calculating the absorption ratio of 280 nm/385 nm. The CT tag does not affect biotins complexation with streptavidin and has a minimal quenching effect on fluorescent streptavidin conjugates. ReadiView™ biotin is available in amine-reactive, thiol-reactive, and click-chemistry formats.

Enzyme-Labeled Streptavidin Conjugates

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Enzyme-labeled streptavidin conjugates are commonly used in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunoblotting, and in situ hybridization techniques. Enzymes horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are extensively used as reporter tags because of their high turnover rate, stability, ease of conjugation, and low cost.

To maximize retention of both enzyme and protein activity, our HRP-streptavidin and AP-streptavidin conjugates are prepared at a 1:1 (enzyme to streptavidin) labeling ratio. Chromogenic, fluorogenic, and chemiluminescent substrates are available for these enzyme conjugates.

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Fluorescent Streptavidin Conjugates

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Fluorescent streptavidin conjugates are widely used in fluorescence imaging to detect biotinylated antibodies, ligands and DNA probes for in situ hybridization techniques, immunohistochemistry and multicolor flow cytometry. We offer a comprehensive portfolio of fluorescent streptavidin labeled with iFluor® dyes, mFluor™ dyes, phycobiliproteins, tandem dyes, and more. Benefits of our iFluor® streptavidin conjugates include exceptionally bright fluorescence, excellent photostability, and pH insensitive fluorescence over a wide molar range.

iFluor® Streptavidin Conjugates

Streptavidin conjugates labeled with iFluor® dyes exhibit exceptionally bright and photostable fluorescence, outperforming Alexa Fluor® and other spectrally similar conjugates. iFluor® streptavidin conjugates are designed for various cell analysis and protein analysis applications, including imaging, flow cytometry, and fluorescent western blotting. As a precaution, avoid using blue fluorescent streptavidin conjugates when detecting low abundance targets. Blue fluorescent dyes have lower fluorescence and can experience a higher non-specific background than other dyes.

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1. Ext. Coeff. = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm^-1M^-1.
2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. N/D = Not determined.

mFluor™ Streptavidin Conjugates for Flow Cytometry

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Streptavidin conjugates labeled with mFluor™ dyes exhibit minimal self-quenching, producing intensely fluorescent and photostable conjugates for flow cytometry. mFluor™ streptavidin conjugates are designed for optimal excitation by one of the major laser lines commonly equipped in flow cytometers, such as the 405 nm, 488 nm, 532 nm, 561 nm, or 633 nm laser lines.

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Streptavidin APC, PE, PerCP and Tandem Dye Conjugates for Flow Cytometry

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Streptavidin PE, APC, and PerCP conjugates are commonly used in applications that require high sensitivity but not photostability, primarily flow cytometry, FACS, immunophenotyping, and microarrays. The unusually bright fluorescence of phycobiliproteins is due to their unusually high extinction coefficients and quantum yields.

Tandem dye conjugates compromise of a donor phycobiliprotein (e.g., APC or PE) labeled to acceptor iFluor® dyes that emit fluorescence at a longer wavelength. Using fluorescence resonance energy transfer (FRET), light absorbed by the donor phycobiliprotein results in fluorescence emission of the acceptor. In flow cytometry, tandem dyes are ideally suited for multicolor parametric analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts.

Streptavidin-Xtra™ Conjugates

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The optimal degree of labeling of Streptavidin-Xtra™ iFluor® conjugates produce the brightest fluorescent streptavidin conjugates for the detection of biotinylated antibodies in cell imaging, flow cytometry, and western blot applications. Streptavidin-Xtra™ iFluor® conjugates image low-abundance targets with greater sensitivity and detail than traditional streptavidin Alexa Fluor® conjugates.

Advantages

• Bright and photostable fluorescence without significant self-quenching
• High signal-to-noise ratios: 3~5 fold signal improvement versus Alexa Fluor®
• pH insensitive fluorescence over a wide molar range

Product Ordering Information

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