Biotinylation reagents

The following sections discuss some of the more common biotinylation reagents available for modification of proteins and other biomolecules. Each biotin derivative contains a reactive portion (or can be made to contain a reactive group) that is specific for coupling to a particular functional group on another molecule. Careful choice of the correct biotinylation reagent can result in directed modification away from active centers or binding sites, and thus preserve the activity of the modified molecule. 

Amine-reactive biotinylation reagents contain reactive groups off biotin s valeric acid side chain that are able to form covalent bonds with primary amines in proteins and other molecules. Two basic types are commonly available N-hydroxysuccinimide (NHS) esters and carboxylates. NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4), 

The only potential deficiency in using D-biotin to modify directly a protein is the relatively short spacer arm afforded by the indigenous valeric acid group. Some applications may require longer spacers to maintain good binding potential toward avidin or streptavidin. 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

Biocytin should not be used in a carbodiimide reaction to modify proteins or other molecules, since it contains both a carboxylate and an amine group. A carbodiimide-mediated reaction, as suggested for D-biotin previously, would cause self-conjugation and polymerization of this reagent. 

The highly specific interaction of avidin with the small vitamin biotin can be a useful tool in designing assay, detection, and targeting systems for biological analytes (see 

Another variable to consider in choosing biotinylation reagents is the use of a biotin analog such as iminobiotin that has a moderated affinity constant in its binding of avidin or streptavidin (Section 3.1). Analogs may be useful if release of the avidin— 

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The biotinylation of amine-dendrimers may be accomplished using either an organic reaction environment or an aqueous medium. For modification of PAMAM dendrimers with a biotinidase resistant biotin compound, Wilbur et al. (1998) performed the reaction in DMF with triethylamine as catalyst (proton acceptor). The following protocol illustrates this type of procedure using the biotinylation reagent NHS-PEG/pbiotin, which closely compares to the biotinidase insensitive compound used in the published procedure. 

Add a 3-fold molar excess of biotinylation reagent over the molar quantity of dendrimer present. For the use of sulfo-NHS-LC-biotin (MW 556), this represents the addition of 2.1pmol or 1.16mg. This reaction ratio will result in a modification level of about 2.5 biotin groups per dendrimer. Other molar ratios also may be used, depending on the desired level of modification and the intended use for the conjugate. 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

Figure 11.1 The basic design of a biotinylation reagent includes the bicyclic rings and valeric acid side chain of D-biotin at one end and a reactive group to couple with target groups at the other end. Spacer groups may be included in the design to extend the biotin group away from modified molecules, thus ensuring better interaction capability with avidin or streptavidin probes.

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

Remove unreacted biotinylation reagent and reaction by-products by gel filtration using a desalting resin or dialysis against PBS. 

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

Dissolve the antibody to be biotinylated in 50 mM sodium bicarbonate, pH 8.5, at a concentration of 10 mg/ml. Other buffers and pH conditions between pH 7 and 9 can be used as long as no amine-containing buffers like Tris are present. Avoid also the presence of disulfide reducing agents that can cleave the disulfide group of the biotinylation reagent. 

Add 0.3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

The reagent is similar to another maleimide-containing biotinylation reagent, 3-(N-maleimi-dopropionyl) biocytin, a compound used to detect sulfhydryl-containing molecules on nitrocellulose blots after SDS-electrophoresis separation (Bayer et al., 1987). Biotin-BMCC should be useful in similar detection procedures. 

Using a similar approach, Clq has been modified with biotin-HPDP and allowed to interact with its specific receptor. Subsequent purification of the Clq receptor was accomplished through cleavage of the disulfide bridge of the biotinylation reagent (Ghebrehiwet et al., 1988). 

Figure 11.10 This biotinylation reagent reacts with sulfhydryl groups through its iodoacetamide end to form thioether bonds.

An analog of this biotinylation reagent with a longer spacer arm also exists. Biotin-LC-hydrazide contains a 6-aminocaproic acid extension off its valeric acid group (Thermo Fisher). 

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

Add a quantity of photobiotin solution to the protein solution to give at least a 5-fold molar excess of biotinylation reagent. 

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