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Avidin-Biotin Interaction

The interaction of biotin and avidin or streptavidin has been exploited for use in many protein and nucleic acid detection and purification methods. Because the biotin label is stable and small, it rarely interferes with the function of labeled molecules enabling the avidin-biotin interaction to be used for the development of robust and highly sensitive assays.

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Protein Methods Library Home

 

 

 

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Overview of Detection Probes

Introduction: The Avidin-Biotin Interaction

Avidin is a protein derived from both avians and amphibians that shows considerable affinity for biotin, a co-factor that plays a role in multiple eukaryotic biological processes. Avidin and other biotin-binding proteins, including streptavidin and NeutrAvidin Protein, have the ability to bind up to four biotin molecules, as shown in the diagram below, making this interaction ideal for both purification and detection strategies.

Diagram of avidin-biotin interaction
Schematic of the avidin-biotin interaction. Avidin, streptavidin or NeutrAvidin Protein can bind up to four biotin molecules, which are normally conjugated to an enzyme, antibody or target protein to form an avidin-biotin complex. † denotes that avidin is also often conjugated to an antibody, target protein or immobilized support

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Avidin-Biotin Handbook

Advantages of using Avidin-Biotin Systems

The avidin-biotin complex is the strongest known non-covalent interaction (Kd = 10-15M) between a protein and ligand. The bond formation between biotin and avidin is very rapid, and once formed, is unaffected by extremes of pH, temperature, organic solvents and other denaturing agents. These features of biotin and avidin – features that are shared by streptavidin and NeutrAvidin Protein – are useful for purifying or detecting proteins conjugated to either component of the interaction.

Applications for which the avidin-biotin interaction is used include:

Like secondary antibodies, the avidin-biotin detection system allows an almost unlimited number of primary detection reagents (i.e., antibodies, nucleic acids probes and ligands) to be easily captured, recovered, immobilized or detected with a very small number of secondary detection reagents generated by modifying avidin or streptavidin. Furthermore, if a specific biotinylated molecule is not available, there are many commercially available reagents to facilitate biotinylation in the lab. Likewise, avidin and streptavidin can also be modified as needed.

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Biotin Labeling Reagents

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Overview of Biotinylation

Disadvantages of using the Avidin-Biotin System

Although the avidin-biotin system is simple to set up and use, it does have certain limitations. Because any biotinylated molecule will bind to any biotin-binding protein, these reagents must be used in combination with other detection-probe systems (i.e., primary-secondary antibodies) for multiplex experiments.

Also, because biotin is a biological molecule, endogenous biotin can cause background and specificity issues when performing assays with certain biotin-rich tissues and extracts (i.e., brain, liver, milk, eggs, corn). This also applies to samples containing endogenous biotin-binding proteins such as eggs (source of avidin) or bacteria like Streptomyces avidinii (source of streptavidin).

For purification applications, the strength of the binding interaction between biotin and avidin is a factor that limits its utility. This is because harsh conditions are required to break the avidin-biotin bonds (i.e., to dissociate and elute), and these may denature target proteins. To overcome this limitation, modified versions of avidin resins and modified forms of biotin labeling reagents are commercially available which make the interaction readily reversible. These include monomeric avidin, cleavable disulfide biotin reagents, and iminobiotin and desthiobiotin derivatives (see discussion of Protein Isolation and Enrichment below).

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Tech Tip#16: Block endogenous biotin

 

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Monomeric Avidin Resins and Kit

Desthiobiotinylation Reagents

Biotin and the Biotin-binding Proteins

Biotin

Biotin is a vitamin (Vitamin H, Vitamin B7, Coenzyme R) that is present in small amounts in all living cells and is critical for a number of biological processes including cell growth and the citric acid cycle. Biotin is abundant in certain plant and animal tissues such as corn kernels, egg yolk, brain, liver and blood. The valeric acid side chain of the biotin molecule can be derivatized in order to incorporate various reactive groups that facilitate the addition of a biotin tag to other molecules. Because biotin is relatively small (244.3 Daltons), it can be conjugated to many proteins and other molecules without significantly altering their biological activity. The highly specific interaction of biotin-binding proteins with biotin make it a useful tool in assay systems designed to detect and target biological analytes.

Chemical structure of biotin
Chemical structure of biotin.

Once biotin is attached to a molecule, the biotin tag can be used to facilitate affinity purification of that molecule using an immobilized biotin-binding protein. Alternatively, a biotinylated molecule can be immobilized through interaction with a biotin-binding protein, and then used to affinity purify other molecules that specifically interact with it (i.e., co-immunoprecipitation or pull-down assays). In the context of immunohistochemistry and immunoblotting, biotin is most often conjugated to primary or secondary antibodies, and the biotin tag is then detected with a biotin-binding protein that is conjugated to an enzyme, fluorophore or other reporter molecule. Many proteins, such as antibodies, can be labeled with several biotin tags, each able to be bound by a biotin-binding protein. An optimized biotin-to-probe ratio can greatly increase the signal output of a detection system making it possible to create very sensitive assays.

Biotin-labeled antibodies and other molecules are readily available from commercial suppliers making assay development routine for many applications. For assays in which a biotinylated probe is not available, there are many biotinylation reagents that enable researchers to chemically label proteins, nucleic acids and surface materials to make custom assay reagents.

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Biotin and Biotin-Conjugated Proteins and Enzymes

Antibody Purification Products

 

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Secondary Antibody Selection Guide

Blocking Strategies for IHC

Avidin-Biotin Complex for IHC

 

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Tech Tip #15: Biotinylate carboxyl groups with EDC and Biotin Hydrazide

Tech Tip #46: Preferentially biotinylate N-terminal alpha-amino groups in peptides

Avidin

Avidin is a biotin-binding protein that is believed to function as an antibiotic in the eggs of birds, reptiles and amphibians. Chicken avidin has a mass of 67,000-68,000 Daltons and is formed from four 128 amino acid-subunits, each binding one molecule of biotin. Avidin is highly glycosylated, with about 10% of its total mass being carbohydrate and contributing to its basic isoelectric point (pI) of 10-10.5 and high solubility in water and aqueous salt solutions. Because avidin is easily purified from chicken egg whites, it is very economical to produce (much more so than streptavidin).

Avidin has a very high affinity for up to four biotin molecules and is stable and functional over a wide range of pH and temperature. Avidin is amenable to extensive chemical modification with little to no effect on function, making it useful for the detection and protein purification of biotinylated molecules in a variety of conditions. The specific interaction of avidin with biotin makes it a useful tool in designing nonradioactive detection systems with excellent sensitivity. The carbohydrate content and basic pI of avidin can result in a high amount of nonspecific binding, so careful optimization of blocking and wash conditions are required to obtain the best assay results when it is used.

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Avidin Protein and Conjugates

Streptavidin

Streptavidin is a tetrameric biotin-binding protein that is isolated from Streptomyces avidinii and has a mass of 60,000 Daltons. While avidin and streptavidin have very little amino acid homology, their structures are very similar. Like avidin, streptavidin is thought to function as an antibiotic and has a very high affinity for biotin (Kd = 10-14 to -15M). Unlike avidin, streptavidin has no carbohydrate and has an acidic isoelectric point (pI = 5) that gives streptavidin a significantly lower solubility than avidin. Commercially available streptavidin products, for example Thermo Scientific Pierce Streptavidin, may be a recombinant form of streptavidin with a mass of 53,000 Daltons and near-neutral isoelectric point (pI = 6.8 to 7.5).

Streptavidin's lack of glycosylation and lower pI results in a lower degree of nonspecific binding, especially lectin binding, compared to that observed for avidin in immunohistochemistry applications. This makes streptavidin an ideal reagent choice for many detections systems. In contrast, streptavidin contains a bacterial recognition sequence, referred to as an RYD motif, which is similar to the mammalian RGD motif and can bind to cell surface receptors causing background signal in certain assay samples. Also, both native and recombinant streptavidin are more expensive to produce, making them more costly than avidin-based assay reagents.

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Pierce Streptavidin and Conjugates

Pierce High Sensitivity Streptavidin HRP Conjugate

Pierce Streptavidin Poly-HRP Conjugate

Streptavidin DyLight Fluorescent Conjugates

Deglycosylated Avidin (NeutrAvidin)

Both avidin and streptavidin have major advantages and disadvantages. The key advantages of avidin are low cost of production and very high solubility, and the key disadvantages are high pI and a high propensity for nonspecific interactions and lectin binding. Streptavidin has lower nonspecific binding and a near neutral pI. However, streptavidin is more costly to produce, and the RYG sequence does create specificity issues in certain applications. A much more ideal reagent would not have the nonspecific binding issues of either avidin or streptavidin, have a near neutral pI and be cost effective to produce. This is accomplished with deglycosylated avidin.

Deglycosylated avidin, commercially available as Thermo Scientific NeutrAvidin Protein, avoids the major drawbacks of both native avidin and streptavidin. NeutrAvidin Protein (60,000 Daltons) has a reduced mass compared to avidin (67,000 Daltons) and retains its high biotin-binding affinity. Deglycosylation of avidin reduces lectin binding to undetectable levels and lowers the isoelectric point (pI=6.3), effectively eliminating the major causes of nonspecific binding for avidin. Because lysine residues remain available, NeutrAvidin Protein can be derivatized or conjugated as easily as streptavidin but lacks the RYD sequence that can cause nonspecific binding in IHC assays. Together, the high biotin-binding affinity and low nonspecific binding make NeutrAvidin Protein the most ideal biotin-binding protein.

Properties of biotin-binding proteins.
  Avidin
(chicken)
Streptavidin
(recombinant)
NeutrAvidin
(from avidin)
Molecular Weight (kDa) 67 53 60
Biotin-binding Sites 4 4 4
Isoelectric Point (pI) 10 6.8 to 7.5 6.3
Specificity low high highest
Affinity for Biotin (Kd) ~10-15M ~10-14 to -15M ~10-15M
Nonspecific Binding high low lowest

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NeutrAvidin Protein and Conjugates

Pierce High Sensitivity NeutrAvidin HRP Conjugates

NeutrAvidin Protein DyLight Fluorescent Conjugates

Applications for Avidin-Biotin Probes

The avidin-biotin system can be used for numerous laboratory methods. The most common methods use avidin or streptavidin for the detection of biotinylated probes. The following is an overview of some of the major laboratory methods effectively using this system.

 

Protein Detection

Protein detection is a core laboratory method used to monitor protein purification, production, expression levels, etc. Because most proteins are not easily distinguish from other proteins in a complex sample, antibodies and other target-specific probes are used to facilitate indirect detection of specific proteins. Using the avidin-biotin systems, biotinylated primary or secondary antibodies can be detected with a biotin-binding protein conjugated to either fluorescent or enzymatic detection reagents. Common assays using this format include IHC, Western blotting and ELISA.

A key benefit of using avidin-biotin systems for protein detection is the ability to amplify the original protien signal to improve detection of proteins expressed at low levels by forming large avidin-biotin complexes, as diagrammed below. These complexes enrich the conjugated reporter (fluorophore or enzyme) to the site of the target antigen for greater signal detection.

Diagram of signal amplification by avidin biotin complex formation
Schematic of signal amplification by avidin-biotin complex formation.

IHC, Western blotting and ELISA share the common procedure of detecting a target protein that is immobilized in situ or in vitro. In each assay, target protein(s) are immobilized by fixation, adsorbed or captured on a surface and then probed with a biotinylated antibody. The biotin tags on the antibody are then used to localize a biotin-binding protein conjugated to a detection reagent.

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Overview of IHC

Overview of Western blotting

Overview of ELISA

Nucleic Acid Detection

Nucleic acid detection is another core laboratory method used to determine the presence, abundance or sequence changes in specific polynucleotides. While many laboratories have switched to more rapid forms of detection, such as real-time polymerase chain reaction (RT-PCR), Northern and Southern blotting remain effective methods for confirming the presence of specific DNA and RNA sequences as well as their size and integrity. While not as rapid as PCR methods, this technique is also valuable for the in situ hybridization to bacterial colonies and bacteriophage plaques when performing large cloning screens.

For these methods, a target nucleotide fragment is transferred to and immobilized on a blotting membrane. Following a stringent blocking procedure, the target sequence is then hybridized with a biotinylated complimentary nucleotide probe. The biotin tags can then be localized and detected using a biotin-binding protein conjugated to a detection reagent.

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DNA & RNA Labeling

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LightShift Chemiluminescent EMSA Kit

LightShift Chemiluminescent RNA EMSA Kit

North2South Chemiluminescent Detection Kit

Protein Isolation and Enrichment

Immunoprecipitation (IP) assays can be simplified and enhanced by using avidin-biotin strategies. In some procedures, the antibody is added directly to the assay sample to initiate binding, followed by capture of the antibody-target complex using Protein A agarose or a similar support. In other procedures, the antibody is first immobilized and then used to capture the target antigen. In either procedure described above, the presence of immunoglobulins in the sample (especially in the later case) can significantly reduce target antigen recovery and potentially increase nonspecific interactions. Furthermore, when the antibody-antigen complex is being captured from a complex sample, long incubation times may be required for supports like Protein A to allow sufficient time for antibody immobilization. These problems can be eliminated by using biotinylated primary antibodies and an immobilized biotin-binding protein. Because biotin-binding proteins will only bind to a biotin tag, endogenous immunoglobulins are not a source for concern. Furthermore, because of the very strong affinity for biotin, incubation periods for either pre-immobilization of the antibody or the antibody-antigen complex are more consistent, with only target concentration, sample viscosity and volume being major sources of variability.

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Immunoprecipitation (IP)

 

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Biotin Labeling Reagents for Proteins

Avidin-biotin chemistry makes possible many other kinds small-scale purification and enrichment strategies besides immunoprecipitation. For example, membrane-impermeable biotinylation reagents can be used to tag cell surface proteins and then facilitate separation of those proteins from all other cellular proteins. Using more advanced chemistry, the biotin tags can be used to specifically enrich specific types of proteins such as ATPases, GTPases and serine hydrolases. Such experiments are not feasible using only antibodies, especially as a method to discover novel proteins.

However, these enrichment strategies (where the goal is to elute and recover the biotin-labeled target from the streptavidin resin) typically require adaptations of the standard avidin-biotin system. This is because the native interaction is very strong and highly resistant to dissociation. Harsh, denaturing conditions (8 M guanidine•HCl, pH 1.5 or boiling in SDS-sample loading buffer) are required to efficiently dissociate avidin:biotin complexes. Such conditions damage the support irreversibly so that it cannot be reused, and denatures the eluted proteins so that they do not maintain any biological activity.

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Cell Surface Protein Isolation Kit

Active-Site Probes and Enrichment Kits

Modifications of avidin-biotin chemistry that make elution possible:

  1. Monomeric avidin resin: Unlike the native tetrameric protein, monomeric avidin subunits bind less strongly to biotin, allowing competitive displacement of captured biotinylated targets using excess free biotin.
  2. Cleavable biotinylation reagents: Certain biotinylation reagents contain a disulfide bond in their spacer arms or conjugate to protein via disulfide bonds. Biotinylated proteins can be eluted from the affinity resin by cleaving the disulfide bond with reducing agent. The biotin group is left bound to the resin, and the protein is recovered (free of any biotin label). The Cell Surface Protein Isolation Kit uses this strategy (labeling with Sulfo-NHS-SS-Biotin).
  3. Desthiobiotin and iminobiotin derivatives: These are modified forms of biotin that bind less strongly to avidin and streptavidin proteins. The Active-Site Probes and Enrichment Kits use desthiobiotin reagents. Several desthiobiotinylation reagents are available for labeling proteins at different functional groups.

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Monomeric Avidin Resins and Kit

Sulfo-NHS-SS-Biotin

Desthiobiotinylation Reagents

Protein Interactions

Similar to immunoprecipitation, traditional co-immunoprecipitation assays require a specific primary antibody for capturing a target antigen. In the co-immunoprecipitation, the target antigen is often referred to as the "bait" protein and the ultimate goal is to use the bait protein to recover additional interacting "prey" proteins. Like immunoprecipitation assays, a biotinylated capture antibody can help overcome potential issues associated with the capture and enrichment process. However, the co-immunoprecipitation assay can be further modified so that no capture antibody is needed. The resulting pull-down assay is typically performed with a tagged bait protein. In many applications, the tagged bait protein is a recombinant fusion protein modified with one of several affinity tags (6x histidine, hemagglutinin antigen, glutathione s-transferase, etc.), but it is also possible to perform these assays with a biotinylated bait protein. The bait protein simply needs to be modified with a biotin tag prior to performing the pull-down assay. While requiring a source of purified bait protein, pull-down assays performed in this manner overcome potential issues that can arise when recombinant modifications generate inactive protein or other undesirable artifacts.

The bait for a pull-down assay does not necessarily need to be a protein. Peptides, drugs and nucleic acids may also be used as the bait for capturing interacting proteins. These probes can also be used to detect protein interaction without the capture and enrichment step. In the EMSA or gel shift assay, nucleic acid-binding proteins are detectable because they cause a change in the migration rate of labeled probes during electrophoresis through a gel matrix. Biotinylated probes used in this manner are quite effective when used with an optimized detection system.

Another advanced method for detecting protein interactions is the label transfer technique. In this procedure, a bait protein is modified with a transferable tag. Reagents designed for this purpose have two reactive groups, the first of which is reacted with the bait protein and the second reactive group is activated after a bait-prey protein interaction is established. The when the tag is biotin, these label transfer reagents enable capture and enrichment of the interacting target protein for identification in subsequent steps.

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Overview of Protein-Protein Interaction Analysis

Overview of Protein-Nucleic Acid Interactions

Label Transfer Protein Interaction Analysis

 

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Pierce Biotinylated Protein Interaction Pull-Down Kit

Amine-directed Biotin Label Transfer Reagent and Kit

Sulfhydryl-directed Biotin Label Transfer Reagents

References

  1. Green N.M. (1963). Avidin. 3. The Nature of the Biotin-Binding Site. Biochem J. 89:599–609.
  2. Livnah, O; Bayer, EA; Wilchek, M; Sussman, JL (1993). "Three-dimensional structures of avidin and the avidin-biotin complex". Proceedings of the National Academy of Sciences of the United States of America. 90(11): 5076–80.
  3. Argarana C.E. et. al., (1986). Molecular cloning and nucleotide sequence of the streptavidin gene. NAR. 14(4):1897–1882.
  4. Marttila, A.T. et. al., (2000). Recombinant NeutraLite Avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties. FEBS Letters. 467(1):31–36.
 
Written and/or reviewed by Jared Snider, Ph.D.

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