Biotin labeling

Fig. 34 Functionalisation of bait peptides. I Gold-labelled streptavidin is attached directly. 2 Biotin-labelled anti-FLAG antibody is bound to the FLAG on the peptide (reaction b) and this is used to attach gold-labelled streptavidin (reaction d). Reprinted with permission from Ryadnov and Woolfson [76]. Copyright 2004 American Chemical Society...

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Dirks RW, van Gijlswijk RP, Tullis RH et al (1990) Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides. J Flistochem Cytochem 38 467 173... 

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

Manz, B., Heubner, A., Kohler, I., Grill, H. J., and Pollow, K. (1983). Synthesis of biotin-labeled dexamethasone derivatives. Novel hormone-affinity probes. Eur.J. Biochem. 131,... 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

In a similar way, the use of oligonucleotide-immobilized beads enabled the realization of DNA sensitive biochips that could be used to detect biotin labelled sequence as 5.108 molecules59. 

Figure 6.2 The trifunctional reagent sulfo-SBED reacts with amine-containing bait proteins via its NHS ester side chain. Subsequent interaction with a protein sample and exposure to UV light can cause crosslink formation with a second interacting protein. The biotin portion provides purification or labeling capability using avidin or streptavidin reagents. The disulfide bond on the NHS ester arm provides cleavability using disulfide reductants, which effectively transfers the biotin label to an unknown interacting protein.

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS group, which forms a disulfide linkage after reaction. The modified protein then is allowed to interact with a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting proteins. Cleavage of the disulfide bond effectively transfers the biotin label to the unknown interacting protein.

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but instead of transferring a radioactive label, it contains a fluorescent portion that is transferred to a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein interaction. Cleavage of a disulfide bridge after capture of interacting proteins results in transfer of a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modification then can be used to detect or isolate the unknown interactor for subsequent identification. 

The psoralen ring system can intercalate within double-stranded DNA or RNA and induce the formation of adducts with adjacent thymine bases (Figure 11.15). The furan-side and pyrone-side of the tricyclic rings in psoralen both can form cycloaddition products with the 5,6-double bond of thymine residues, which results in crosslinks between the DNA strands with a PEG-biotin label sticking out. 

Biotinylated oligosaccharides are convenient probes of carbohydrate interactions, because the biotin label can be captured or detected using an avidin or streptavidin derivative. For instance, immobilized streptavidin can be used to purify glycoconjugates that have been labeled... 

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 18.22 Biotin-PEG -amine can be used to add a biotin label to carboxylate-containing molecules using the EDC/(sulfo)NHS reaction, which forms a stable amide linkage.

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluoro-phore, etc., then a very sensitive detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. in... 

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively couples a biotin label to every 100-200 base residues. The result is an oligonucleotide probe detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA modification are summarized in Chapter 11, Section 4. 

Figure 28.11 Sulfo-SBED is a label transfer agent that contains a water-soluble sulfo-NHS ester to label bait proteins and a phenyl azide group for photoreactive capture of a prey protein. The biotin label can be used for detection or isolation of protein-protein conjugates using (strept)avidin reagents. The stars indicate the atoms that were used to measure the indicated molecular dimensions.

Biotin label transferred to unknown prey protein... 

Figure 28.13 A sulfo-SBED-captured protein interaction can be released using DTT to cleave the disulfide within the cross-bridge leading to the bait protein. The result transfers the biotin label to the unknown interacting protein. The biotin tag thus allows the interacting protein to be detected or isolated using (strept)avidin reagents.

Langer, P.R., Waldrop, A.A., and Ward, D.C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. 

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