Biotin probes

Because the disposal of radioactive waste is becoming increasingly expensive, nonradioactive probes have been developed. One of the most successful is based on the vitamin biotin (see p. 379), which can be chemically coupled to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to... 

Pierce Chemical Company PO Box 117 Rockford, IL 61105 Cross-linking agents, protein modification reagents, fluorescent probes, biotin avidin systems, chromatography supports, kits for production of various conjugates... 

Hybridization Mix. 55% formamide, 10% dextran sulfate, 1 mg/ml blocking (degraded salmon sperm) DNA, 1 fig/ml of labeled probe (biotin-11-dUTP labeled), IX SSC. 

Biotin methyl ester Fluorophore-labelled probes Biotin nitrophenyl ester MeCN Biotin methyl ester [58]... 

Fig. 7 Frequency responses of streptavidin-treated QCMs (10 MFIz) to (1) probe (biotin-terminated 30-mer nucleotide) immobilization, (2) MMO target (upper curve) and MMl target (lower curve) DNA hybridization, and (5) MutS protein binding. Arrows (3), (4), and (6) show the change in buffer system. (From Su et al. [53].)...

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Activity-based protein profiling (ABPP) is a chemical proteomic strategy in which active-site-directed covalent probes are used to profile the functional states of enzymes in complex proteomes. Activity-based probes (ABPs) can distinguish active enzymes from their inactive zymogens or inhibitor-bound forms. They contain a reactive group intended to modify enzyme active sites covalently and a reporter group (typically rhodamine or biotin) that assists in detection and identification of protein targets. 

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,... 

First entry on each line is the tag. Square brackets enclose the probe donor + transfer enzyme (where applicable) or reaction. Probe organic fluorophores, nanoparticles (can be QDs as in methods 2, 3, 8, 9) or a bridging/recognition moiety. If desired, the latter can serve in a second orthogonal reaction ( piggyback strategy). See text. Biotin readout probes linked to avidin, streptavidin, anti-biotin 7[101] ... 

Figure 6.1 The Wedekind trifunctional crosslinker can react with amine groups via its p-nitrophenyl ester to form amide bond linkages. The phenyl azide group then can be photoactivated with UV light to generate covalent bond formation with a second molecule. The biotin side chain provides binding capability with avidin or streptavidin probes.

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

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.

After molecules modified with sulfo-NHS-SS-biotin are allowed to interact with avidin or streptavidin probes, the complexes can be cleaved at the disulfide bridge by treatment with 50 mM DTT. Reduction releases the biotinylated molecule from the avidin or streptavidin capture reagent without breaking the (strept)avidin interaction. The use of disulfide biotinylation reagents... 

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