Biotin group

Figure 7.20 The multivalent surface of dendrimers can be used to couple biotin groups and labels for detection in immunoassays. One such conjugate was made by coupling NHS-biotin and a maleimido-iron chelate to an amine-dendrimer for use in an unique carbonyl metallo assay method.

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. 

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.

Figure 11.7 Sulfo-NHS-SS-biotin reacts with amine groups to form amide bonds. The biotin group can be later cleaved off the modified molecule by reduction of its internal disulfide linkage.

Figure 11.9 Biotin-HPDP reacts with sulfhydryl-containing molecules through its pyridyl disulfide group, forming reversible disulfide bonds. The biotin group may be released from modified molecules by reduction with DTT.

Biotin modification reagents are widely used to attach a biotin group to proteins or other molecules for subsequent use in avidin, streptavidin, or NeutrAvidin separations or assays. Traditional biotin compounds containing aliphatic or other hydrophobic linker arms are discussed in detail in Chapter 11. In this section, the biotin-PEG compounds exclusively are discussed due to their unique hydrophilic properties, which include low nonspecific binding character and low immunogenicity. 

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. 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

NHS ester Chromogenic Hydrophilic PEG3 Biotin group... 

Figure 18.23 Biotin-PEG3-benzophenone is a water-soluble photoreactive biotinylation reagent that can be used to add a biotin group to surfaces or molecules containing no easily derivatized functional groups.

Another popular tag for use with immunoglobulins is biotin. Modification reagents that can add a functional biotin group to proteins, nucleic acids, and other molecules now come in many shapes and reactivities (Chapter 11). Depending on the reactive group present on the biotinylation... 

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.

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Figure 28.10 Trifunctional label transfer agents contain two arms with terminal reactive groups and a third arm with a label or affinity tag, such as a biotin group. One of the reactive groups typically is thermoreactive to couple with bait proteins, while the second reactive group usually is photoreactive. The thermoreactive arm has a cleavable cross-bridge to facilitate release of the captured protein and transfer of the label of affinity tag to it.

---