NHS-LC-biotin

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. 

The NHS ester end of NHS-LC-biotin reacts with amine groups in proteins and other molecules to form stable amide bond derivatives (Figure 11.4). Optimal reaction conditions are at a pH of 7-9, but the higher the pH the greater will be the hydrolysis rate of the ester. Avoid amine-containing buffers which will compete in the acylation reaction. NHS-LC-biotin is insoluble in aqueous reaction conditions and must be solubilized in organic solvent prior to the addition of a small quantity to a buffered reaction. Preparation of concentrated stock solutions may be done in DMF or DMSO. Nonaqueous reactions also may be done with this reagent for the modification of molecules insoluble in water. The molar ratio of NHS-LC-biotin to a... 

Figure 11.4 NHS-LC-biotin provides an extended spacer arm to allow greater distance between the biotin rings and a modified molecule. Reaction with amines forms amide linkages.

NHS-LC-biotin can be used to add a biotin tag to monoclonal antibodies directed at certain tumor antigens. The biotinylated monoclonals are allowed to bind to the tumor cell surfaces in vivo, and subsequent administration of an avidin or streptavidin conjugate can form the basis for inducing cytotoxic effects or creating traceable complexes for use in imaging techniques (Hnatowich et al., 1987). 

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 following protocol is a suggested method for the biotinylation of proteins with either NHS-LC-biotin or sulfo-NHS-LC-biotin. 

Dissolve NHS-LC-biotin (Thermo Fisher) in dry DMF at a concentration of 40 mg/ml. This stock solution is stable for reasonable periods, although long-term storage is not recommended. For use of the water-soluble sulfo-NHS-LC-biotin, a stock solution may be prepared in either organic solvent or water, or the solid reagent may be added directly to the reaction mixture. If a solution in water is made to facilitate the addition of a small quantity of reagent to a reaction, then the solution should be prepared quickly and used immediately to prevent hydrolysis of the NHS ester. Sulfo-NHS-LC-biotin may be dissolved in water at a concentration of 20 mg/ml. 

Add 50 pi of the NHS-LC-biotin solution in DMF to each ml of the protein solution in two aliquots apportioned 10 minutes apart. Alternatively, add a quantity of the sulfo-NHS-biotin solution prepared in water 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 the sulfo-NHS-biotin solution (8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. 

Figure 20.19 Biotinylated antibodies can be formed by reacting NHS-LC-biotin with available amine groups to create amide bonds.

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. 

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. 

Figure 22.19 Biotinylated liposomes may be formed using biotinylated PE. Reaction of NHS-LC-biotin with PE results in amide bond linkages and a long spacer arm terminating in a biotin group.

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Reaction of NHS-LC-Biotin with Diamine-Modified DNA Probes... 

NHS-LC-biotin is an extended spacer arm derivative of biotin containing an amine-reactive NHS ester (Chapter 11, Section 1). The compound is a popular choice for biotinylating a wide... 

Figure 27.10 Biotinylation of oligonucleotides may be done at the 5 -phosphate end using a diamine derivative and reacting with NHS-LC-biotin.

Dissolve NHS-LC-biotin (Thermo Fisher) in DMSO at a concentration of lOmg/ml. Add 50 pi of the biotinylation solution to the oligo solution. Mix well. 

Instead of BNHS the more hydrophilic derivative sulfo-NHS-biotin (M 443.42) or a spacer derivative, e.g., sulfo-NHS-LC-biotin maybe used. 

If NHS-LC-biotin is used as biotinylating reagent, the elution can be performed using 100 mM triethylamine. 

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