Modification biotinylation reagents

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. 

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. 

Dissolve a sulfhydryl-containing protein or other thiol-molecule in a thiol-free buffer within a pH range of 6.5-7.5. The use of 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, works well for this reaction. The concentration of protein should be in the range of l-10mg/ml. Lower concentrations of protein may result in the need to increase the molar excess of biotinylation reagent to obtain an acceptable level of modification. If a... 

However, since many of the traditional biotinylation reagents, such as NHS-LC-biotin contain hydrophobic spacers, their use with amphipathic liposomal constructions may not be entirely appropriate. A better choice may be to use a hydrophilic PEG-based biotin compound that creates a water-soluble biotin modification on the outer aqueous surface of the liposome bilayer. Biotinylation reagents of this type are discussed in Chapter 18, Section 3. 

Although NHS-LC-biotin and sulfo-NHS-LC-biotin are very popular reagents for biotinylation, they both result in hydrophobic aliphatic biotin modifications on proteins and antibodies. Unfortunately, these groups have a tendency to aggregate in aqueous solution and may cause protein precipitation or loss of activity over time. For this reason, the use of more hydrophilic PEG-based biotin compounds of approximately the same spacer length may be a better alternative for maintaining water solubility of modified proteins (Chapter 18). 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

The use of discrete PEG spacers in the construction of biotinylation compounds not only increases the water solubility of the modification reagent itself, but significantly increases the hydrophilicity and stability of proteins modified with them. Even when high modification levels... 

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