Biotinylation
Encyclopedia
In biochemistry
, biotinylation is the process of covalently attaching biotin
to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin (MW = 244.31). Biotin binds to streptavidin
and avidin
with an extremely high affinity and specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated
to a protein of interest, which allows binding of multiple streptavidin
, avidin
or Neutravidin
protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods.
(PEG) can make water-insoluble reagents soluble or increase the solubility of biotinylation reagents that are already soluble to some extent.
molecules are primary amine groups that are present as lysine side chain epsilon-amines and N-terminal α-amines. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility
.
N-hydroxysuccinimide
(NHS) esters have poor solubility in aqueous solution
s. For reactions in aqueous solution, they must first be dissolve
d in an organic
solvent
, then diluted into the aqueous reaction
mixture. The most commonly used organic solvents for this purpose are dimethyl sulfoxide
(DMSO) and dimethyl formamide (DMF), which are compatible with most proteins at low concentrations. Because of the hydrophobicity of NHS-esters, NHS biotinylation reagents can also diffuse through the cell membrane
, meaning that they will biotinylate both internal and external components of a cell
.
Sulfo-NHS esters are more soluble in water and should be dissolved in water just before use because they hydrolyze easily. The water solubility of sulfo-NHS-esters stems from their sulfonate
group on the N-hydroxysuccinimide
ring and eliminates the need to dissolve the reagent in an organic solvent. Sulfo-NHS-esters of biotin also can be used as cell surface biotinylation reagents, because they do not penetrate the cell membrane
.
The chemical reactions of NHS- and sulfo-NHS esters are essentially identical, in that they both react spontaneously with amines to form an amide bond. Because the target for the ester is a deprotonated primary amine, the reaction is favored under basic conditions (above pH 7). Hydrolysis
of the NHS ester is a major competing reaction, and the rate of hydrolysis increases with increasing pH
. NHS- and sulfo-NHS-esters have a half-life
of several hours at pH 7 but only a few minutes at pH 9.
There is some flexibility in the conditions for conjugating NHS-esters to primary amines. Incubation temperatures can range from 4-37°C, pH values in the reaction range from 7-9, and incubation times range from a few minutes to 12 hours. Buffers containing amines (such as Tris
or glycine
) must be avoided, because they compete with the reaction.
groups are less prevalent on most proteins compared to primary amines, sulfhydryl biotinylation is useful when primary amines are located in the regulatory domain(s) of the target protein or when a reduced level of biotinylation is required. Sulfhydryl-reactive groups such as aleimide]s], haloacetyls and pyridyl disulfides, require free sulfhydryl groups for conjugation; disulfide bonds must first be reduced to free up the sulfhydryl groups for biotinylation. If no free sulfhydryl groups are available, lysines can be modified with various thiolation
reagents (Traut's Reagent, SAT(PEG4), SATA and SATP), resulting in the addition of a free sulfhydryl. Sulfhydryl biotinylation is performed at a slightly lower pH (6.5-7.5) than labeling with NHS esters.
Besides whole proteins, biotinylated peptides can be synthesized by introducing a cysteine (Cys)
residue during synthesis at the terminus of the amino acid chain to get a site specific and oriented biotinylation. Nucleotides can also be biotinylated by incorporation of biotinylated nucleotides.
are found on the C-terminal ends of proteins and on glutamate and aspartate amino acid side chains. Biotinylation reagents that target carboxyl groups do not have a carboxyl-reactive moiety per se but instead rely on a carbodiimide
crosslinker such as EDC
to bind the primary amine on the biotinylation reagents to the carboxyl group on the target protein.
Biotinylation at carboxyl groups occur at pH 4.5-5.5. To prevent crossreactivity of the crosslinker with buffer constituents, buffers should not contain primary amines (e.g., Tris
, glycine
) or carboxyls (e.g., acetate
, citrate
); MES
buffer is an ideal choice.
residues to aldehydes, which then react with hydrazine
- or alkoxyamine-based biotinylation reagents. Sodium periodate
oxidizes the sialic acids on glycoproteins to aldehydes to form these stable linkages at pH 4-6.
Polyclonal antibodies are heavily glycosylated, and because glycosylation does not interfere with the antibody activity, biotinylating the glycosyl groups is an ideal strategy to generate biotinylated antibodies.
s are readily biotinylated in the course of oligonucleotide synthesis
by the phosphoramidite method using commercial biotin phosphoramidite. Upon the standard deprotection, the conjugates obtained can be purified using reverse-phase or anion-exchange HPLC
Photoactivatable biotinylation reagents can also be used to activate biotinylation at specific times in an experiment or during certain reaction conditions, by simply exposing the reaction to UV light at the specific time or condition.
together with a column that has avidin
(also streptavidin
or Neutravidin
) bound to it, which is the natural ligand for biotin. However, harsh conditions (e.g., 6M GuHCl at pH 1.5) are needed to break the avidin/streptavidin - biotin interaction, which will most likely denature the protein carrying the biotin tag. If isolation of the tagged protein is needed, it is better to tag the protein with iminobiotin. This biotin analogue gives strong binding to avidin/streptavidin at alkaline pH, but the affinity is reduced upon lowering the pH. Therefore, a iminobiotin-tagged, functional protein can be released from an avidin/streptavidin column by decreasing the pH (to around pH 4).
or avidin/streptavidin-tagged detection strategies such as enzyme reporters (e.g., horseradish peroxidase
, alkaline phosphatase
) or fluorescent probes
. This can be useful in localization, ELISA
assays, ELISPOT
assays, western blot
s and other immunoanalytical methods.
. This very tight binding makes labeling proteins with biotin a useful tool for applications such as affinity chromatography
using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals. Biotinylated protein such as biotinylated bovine serum albumin (BSA) is used in solid-phase assays as a coating on the well surface in multiwell assay plates. Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity.
Overview of Biotinylation - Includes additional information and figures of reactive groups, biotin and linker regions.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
, biotinylation is the process of covalently attaching biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin (MW = 244.31). Biotin binds to streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
and avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
with an extremely high affinity and specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated
Cross-link
Cross-links are bonds that link one polymer chain to another. They can be covalent bonds or ionic bonds. "Polymer chains" can refer to synthetic polymers or natural polymers . When the term "cross-linking" is used in the synthetic polymer science field, it usually refers to the use of...
to a protein of interest, which allows binding of multiple streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
, avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
or Neutravidin
NeutrAvidin
NeutrAvidin protein is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity...
protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods.
Labeling methods
Proteins can be biotinylated chemically or enzymatically. Chemical biotinylation utilises various conjugation chemistries to yield nonspecific biotinylation of amines, carboxylates, sulfhydryls and carbohydrates (e.g., NHS-coupling gives biotinylation of any primary amines in the protein). Enzymatic biotinylation results in biotinylation of a specific lysine within a certain sequence by a bacterial biotin ligase. Most biotinylation reagents consist of a reactive group attached via a linker to the valeric acid side chain of biotin. As the biotin binding pocket in avidin / streptavidin is buried beneath the protein surface, biotinylation reagents possessing a longer linker are desirable, as they enable the biotin molecule to be more accessible to binding avidin/streptavidin/Neutravidin protein. This linker can also mediate the solubility of biotinylation reagents; linkers that incorporate poly(ethylene) glycolPolyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG) can make water-insoluble reagents soluble or increase the solubility of biotinylation reagents that are already soluble to some extent.
Primary amine biotinylation
The most common targets for modifying proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
molecules are primary amine groups that are present as lysine side chain epsilon-amines and N-terminal α-amines. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility
Solubility
Solubility is the property of a solid, liquid, or gaseous chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. The solubility of a substance fundamentally depends on the used solvent as well as on...
.
N-hydroxysuccinimide
N-Hydroxysuccinimide
N-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
(NHS) esters have poor solubility in aqueous solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
s. For reactions in aqueous solution, they must first be dissolve
Dissolution (chemistry)
Dissolution is the process by which a solid, liquid or gas forms a solution in a solvent. In solids this can be explained as the breakdown of the crystal lattice into individual ions, atoms or molecules and their transport into the solvent. For liquids and gases, the molecules must be compatible...
d in an organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
, then diluted into the aqueous reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
mixture. The most commonly used organic solvents for this purpose are dimethyl sulfoxide
Dimethyl sulfoxide
Dimethyl sulfoxide is an organosulfur compound with the formula 2SO. This colorless liquid is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water...
(DMSO) and dimethyl formamide (DMF), which are compatible with most proteins at low concentrations. Because of the hydrophobicity of NHS-esters, NHS biotinylation reagents can also diffuse through the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
, meaning that they will biotinylate both internal and external components of a cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
.
Sulfo-NHS esters are more soluble in water and should be dissolved in water just before use because they hydrolyze easily. The water solubility of sulfo-NHS-esters stems from their sulfonate
Sulfonate
A sulfonate is a salt or ester of a sulfonic acid. It contains the functional group R-SO2O-.- Sulfonate salts:Anions with the general formula RSO2O− are called sulfonates. They are the conjugate bases of sulfonic acids with formula RSO2OH. As sulfonic acids tend to be strong acids, the...
group on the N-hydroxysuccinimide
N-Hydroxysuccinimide
N-Hydroxysuccinimide is a compound with a molecular weight of 115.09 and a melting point of 95 °C.As it is slightly acidic, it is an irritant to skin, eyes and mucous membranes....
ring and eliminates the need to dissolve the reagent in an organic solvent. Sulfo-NHS-esters of biotin also can be used as cell surface biotinylation reagents, because they do not penetrate the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
.
The chemical reactions of NHS- and sulfo-NHS esters are essentially identical, in that they both react spontaneously with amines to form an amide bond. Because the target for the ester is a deprotonated primary amine, the reaction is favored under basic conditions (above pH 7). Hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of the NHS ester is a major competing reaction, and the rate of hydrolysis increases with increasing pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
. NHS- and sulfo-NHS-esters have a half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...
of several hours at pH 7 but only a few minutes at pH 9.
There is some flexibility in the conditions for conjugating NHS-esters to primary amines. Incubation temperatures can range from 4-37°C, pH values in the reaction range from 7-9, and incubation times range from a few minutes to 12 hours. Buffers containing amines (such as Tris
Tris
Tris is an abbreviation of the organic compound known as trisaminomethane, with the formula 3CNH2. Tris is extensively used in biochemistry and molecular biology. In biochemistry, tris is widely used as a component of buffer solutions, such as in TAE and TBE buffer, especially for solutions of...
or glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
) must be avoided, because they compete with the reaction.
Sulfhydryl biotinylation
An alternative to primary amine biotinylation is to label sulfhydryl groups with biotin. Because free sulfhydrylThiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
groups are less prevalent on most proteins compared to primary amines, sulfhydryl biotinylation is useful when primary amines are located in the regulatory domain(s) of the target protein or when a reduced level of biotinylation is required. Sulfhydryl-reactive groups such as aleimide]s], haloacetyls and pyridyl disulfides, require free sulfhydryl groups for conjugation; disulfide bonds must first be reduced to free up the sulfhydryl groups for biotinylation. If no free sulfhydryl groups are available, lysines can be modified with various thiolation
Thiol
In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
reagents (Traut's Reagent, SAT(PEG4), SATA and SATP), resulting in the addition of a free sulfhydryl. Sulfhydryl biotinylation is performed at a slightly lower pH (6.5-7.5) than labeling with NHS esters.
Besides whole proteins, biotinylated peptides can be synthesized by introducing a cysteine (Cys)
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...
residue during synthesis at the terminus of the amino acid chain to get a site specific and oriented biotinylation. Nucleotides can also be biotinylated by incorporation of biotinylated nucleotides.
Carboxyl biotinylation
Carboxyl groupsCarboxylic acid
Carboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group...
are found on the C-terminal ends of proteins and on glutamate and aspartate amino acid side chains. Biotinylation reagents that target carboxyl groups do not have a carboxyl-reactive moiety per se but instead rely on a carbodiimide
Carbodiimide
A carbodiimide or a methanediimine is a functional group consisting of the formula RN=C=NR. Carbodiimides hydrolyze to form ureas, which makes them uncommon in nature.-Carbodiimide formation:...
crosslinker such as EDC
EDC
EDC may refer to:*Electrodesiccation and curettage, a surgical method for the removal of skin cancers*Eau de Cologne , the oldest perfume classification....
to bind the primary amine on the biotinylation reagents to the carboxyl group on the target protein.
Biotinylation at carboxyl groups occur at pH 4.5-5.5. To prevent crossreactivity of the crosslinker with buffer constituents, buffers should not contain primary amines (e.g., Tris
Tris
Tris is an abbreviation of the organic compound known as trisaminomethane, with the formula 3CNH2. Tris is extensively used in biochemistry and molecular biology. In biochemistry, tris is widely used as a component of buffer solutions, such as in TAE and TBE buffer, especially for solutions of...
, glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
) or carboxyls (e.g., acetate
Acetate
An acetate is a derivative of acetic acid. This term includes salts and esters, as well as the anion found in solution. Most of the approximately 5 billion kilograms of acetic acid produced annually in industry are used in the production of acetates, which usually take the form of polymers. In...
, citrate
Citrate
A citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate.-Other citric acid ions:...
); MES
MES (buffer)
MES is the common name for the compound 2-ethanesulfonic acid. Its chemical structure contains a morpholine ring. It has a molecular weight of 195.2 and the chemical formula is C6H13NO4S...
buffer is an ideal choice.
Glycoprotein biotinylation
Glycoproteins can be biotinylated by modifying the carbohydrateCarbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
residues to aldehydes, which then react with hydrazine
Hydrazine
Hydrazine is an inorganic compound with the formula N2H4. It is a colourless flammable liquid with an ammonia-like odor. Hydrazine is highly toxic and dangerously unstable unless handled in solution. Approximately 260,000 tons are manufactured annually...
- or alkoxyamine-based biotinylation reagents. Sodium periodate
Sodium periodate
Sodium periodate is the sodium salt of periodic acid. It can refer to two different chemical compounds, sodium metaperiodate , which has the formula NaIO4, and sodium orthoperiodate , which has the formula Na2H3IO6...
oxidizes the sialic acids on glycoproteins to aldehydes to form these stable linkages at pH 4-6.
Polyclonal antibodies are heavily glycosylated, and because glycosylation does not interfere with the antibody activity, biotinylating the glycosyl groups is an ideal strategy to generate biotinylated antibodies.
Oligonucleotide biotinylation
OligonucleotideOligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s are readily biotinylated in the course of oligonucleotide synthesis
Oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
by the phosphoramidite method using commercial biotin phosphoramidite. Upon the standard deprotection, the conjugates obtained can be purified using reverse-phase or anion-exchange HPLC
Non-specific biotinylation
Photoactivatable biotinylation reagents are ideal when primary amines, sulfhydryls, carboxyls and carbohydrates are not available for labeling. These reagents rely on aryl azides, which become activated by ultraviolet light (UV; >350nm), which then react at C-H and N-H bonds. Because these types of bonds occur independent of the type of amino acid, this type of biotinylation is termed "non-specific".Photoactivatable biotinylation reagents can also be used to activate biotinylation at specific times in an experiment or during certain reaction conditions, by simply exposing the reaction to UV light at the specific time or condition.
Purification
The biotin tag can be used in affinity chromatographyAffinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
together with a column that has avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
(also streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
or Neutravidin
NeutrAvidin
NeutrAvidin protein is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity...
) bound to it, which is the natural ligand for biotin. However, harsh conditions (e.g., 6M GuHCl at pH 1.5) are needed to break the avidin/streptavidin - biotin interaction, which will most likely denature the protein carrying the biotin tag. If isolation of the tagged protein is needed, it is better to tag the protein with iminobiotin. This biotin analogue gives strong binding to avidin/streptavidin at alkaline pH, but the affinity is reduced upon lowering the pH. Therefore, a iminobiotin-tagged, functional protein can be released from an avidin/streptavidin column by decreasing the pH (to around pH 4).
Detection
This tag can also be used in detection of the protein via anti-biotin antibodiesAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
or avidin/streptavidin-tagged detection strategies such as enzyme reporters (e.g., horseradish peroxidase
Horseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
, alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
) or fluorescent probes
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
. This can be useful in localization, ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
assays, ELISPOT
ELISPOT
The Enzyme-linked immunosorbent spot assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983....
assays, western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
s and other immunoanalytical methods.
Other uses
The non-covalent bond formed between biotin and avidin or streptavidin has a binding affinity that is higher than most antigen and antibody bonds and approaches the strength of a covalent bondCovalent bond
A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
. This very tight binding makes labeling proteins with biotin a useful tool for applications such as affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals. Biotinylated protein such as biotinylated bovine serum albumin (BSA) is used in solid-phase assays as a coating on the well surface in multiwell assay plates. Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity.
Determining the extent of biotinylation
Reaction conditions for biotinylation are chosen so that the target molecule (e.g., an antibody) is labeled with sufficient biotin molecules to purify or detect the molecule, but not so much that the biotin interferes with the function of the molecule. The HABA dye(2-(4-hydroxyazobenzene) benzoic acid) method is used to determine the extent of biotinylation. HABA dye is bound to avidin and yields a characteristic absorbance. When biotinylated proteins or other molecules are introduced, the biotin displaces the dye, resulting in a change in absorbance at 500 nm. This change is directly proportional to the level of biotin in the sample.Further reading
Hermanson, G.T. Bioconjugate Techniques. Academic Press ISBN 0-12-342336-8Overview of Biotinylation - Includes additional information and figures of reactive groups, biotin and linker regions.