Biotin-PEG-Amine Compounds

The following protocol can be used to biotinylate carboxylate-containing molecules in aqueous solution using the EDC/sulfo-NHS reaction. 

Dissolve the biotin-PEG -amine reagent in reaction buffer at a concentration of 25 mM. 

Add a quantity of the biotin-PEG -amine solution to the solution containing the car-boxylate molecule to achieve the desired molar excess. For molecules containing a single carboxylate to be modified, a 1.5- to 2-fold molar excess may be sufficient. However, for proteins or peptides that also contain competing amines, a much larger excess of biotin compound should be used (e.g., 100-fold excess). For instance, for protein biotinylation, add 120 pi of the biotin-PEG -amine solution per ml of the solution prepared in Step 1. 

Immediately before use, dissolve EDC in reaction buffer at a concentration of 25 mM. Add 12 pi of this solution per ml of the combined solution from Step 2. Mix well. 

React for 2 hours at room temperature or 4 hours at 4°C with gentle mixing. 

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Dissolve an amine-containing ligand in 0.1 M sodium carbonate, pFI 9.5. If coupling a small ligand, such as a biotin-PEG-amine compound (Chapter 18), then use a concentration of about 5-10 mM in the carbonate buffer. For proteins, concentrations of 10 nM to 1 pM can be used with success. 

Figure 18.21 Biotin-PEG -amine compounds can be used to modify carboxylate- or aldehyde-containing compounds using a carbodiimide reaction.

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. 

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

Figure 18.15 NHS-chromogenic-PEG3-biotin contains an amine-reactive NHS ester that can be used to label biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of the reagent allows the degree of biotinylation to be quantified by measuring its absorbance at 354 nm. The compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

Biotin compounds containing a PEG spacer that terminates in a primary amine can be used for the labeling of carboxylate molecules (Figure 18.21). Activated carboxylates, such as those... 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

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