Biotin moiety

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

Fig. 8.20 Experimental sequence of surface sensing (a) The sensor surfaces are functionalized with biotinylated BSA (b) Streptavidin is specifically bound to the sensor surface with the biotin moiety (c) Biotin is captured on the sensor surfaces by the streptavidin...

CNTs can be functionalized with protein via non-covalent bond (Li et al., 2005 Kim et al., 2003 Mitchell et al., 2002). For example, (beta-lactamase I, that can be immobilized inside or outside CNTs, doesn t change enzyme s activity (Vinuesa and Goodnow, 2002). Taq enzyme can attach to the outside of CNT, and doesn t change its activity (Cui et al., 2004). Peptide with Histidine and Tryptophan can have selective affinity for CNT(Guo et al., 1998). Monoclonal antibody can attach to SWNTs. Protein-modified CNTs can be used to improve its biocompatibility and biomolecular recognition capabilities (Um et al., 2006). For example, CNTs functionalized with PEG and Triton X-100 can prevent nonspecific binding of protein and CNTs. Biotin moiety is attached to the PEG chains Streptavidin can bind specifically with biotin-CNT (Shim et al., 2002). 

Figure 8.9. (a) Schematic diagram to illustrate the location of the binding sites on avidin and streptavidin molecules. (b) A lipid with a biotin head group. The biotin moiety is at the right of the diagram and locates in the cavities shown in a schematic manner in (a). 

Enzyme labelling of the detection antibody fill the reservoirs with 15 pL of solution of streptavidin labelled with alkaline phosphatase. Incubate this solution in the micro-channels using 10 multi-loadings so as to ensure the coupling of the biotin moiety of the detection antibody with the ALP-labelled streptavidin. Remove the solution in excess at the end of the pumping process. 

Fig. 4. Schematic of a single-step array fabrication process for in vivo biotinylated proteins. Step a A cmde lysate containing the desired biotinylated recombinant protein is printed onto a streptavidin-coated surface coderivatized with a polymer that resists nonspecific protein absorption. Step b Unbound proteins are washed away to leave the purified recombinant protein, specifically immobilized and oriented on the array surface via the biotin moiety on the BCCP tag.

In detail, viral wild-type particles of one set were labeled with the fluorescent dye AlexaFluor (AF) 488 and biotin both groups were attached to surface available lysines. Another batch was labeled with AF568 and biotin, also at addressable lysines. Both types of building block, in the following referred to as CPMV-biotin-AF488 and CPMV-biotin-AF568, were characterized by TEM, UV-visible spectroscopy, native gel electrophoresis, and dot blot studies. TEM studies verified that the particles remained intact after chemical modification. UV-visible spectroscopy confirmed covalent modification and also allowed quantification of the number of labels per particle the particles displayed around 40 biotin moieties and around 200 dyes. 

Lamos and colleagues reported the modification of cyclophilin A (CypA) by binding immunosuppressive cyclosporin A with a benzophenone-Dj j and a biotin moiety (compound 89, Fig. 11a) [91]. As a proof of principle, they used a 1 1 mixture of this TIP and its nondeuterated isoster for the selective PAL and pulldown of CypA among three other proteins. Subsequent tryptic digestion of the elutes and LC-MS/MS analysis allowed the identification of 11 CypA characteristic peptides, two of which were modified with the probe, as evidenced from the double, 11 Da separated, peaks in the mass spectra. The large 11-Da mass difference allowed easy visual recognition of labeled peptides in the mass spectra, which makes this a powerful method for determination of the modification site after PAL however, application in more complex systems still remains to be done. 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

Fig. 9. Avidity controlled interaction kinetics between the anti-biotin antibody 2F5 and the surface-confined biotin moieties. (A) Normalized SPR curves of the 2F5 association/dissociation process on surfaces with relatively high biotin densities. (B) Fluorescence kinetic curves of the 2F5 association/dissociation on surfaces with lower biotin densities.

Four luminescent cyclometallated iridium(III) diimine complexes [Ir(ppy-spacer-biotin)2(NAN)]+ (NAN = Me4-phen, Ph2-phen) (38), each containing two biotin units, have been synthesised and characterised by Lo and co-workers [79], Photoexcitation of these iridium(III) diimine bis(biotin) complexes in fluid solutions at 298 K and in alcohol glass at 77 K results in intense and long-lived 3MLCT (djr(Ir) — ti (NaN))/3IL (ir —> ji ) (Me4-phen) emission. HABA assays and emission titrations indicate that the two biotin moieties of each complex are functional. RET-based emission-quenching experiments, microscopy studies using avidin-conjugated microspheres, and HPLC analysis all reveal that the complexes with a... 

Fig. 3 d Photocleavable biotin derivative made up of a biotin moiety linked through a spacer arm (6-aminocaprioic acid) to allow binding to streptavidin and an alpha-substituted 2-nitrobenzyl nucleus with a iV-hydroxysuccinimidyl reactive group (NHS carbonate) that reacts with primary amines under mild conditions (pH 8) (Taken from [75])... 

Figure 4 Chemical tools for the study of y-secretase. Transition-state anaiog inhibitors inciude hydroxyi-containing moieties that interact with the catalytic aspartates of aspartyl proteases. Helical peptides mimic the APR transmembrane domain and interact with the substrate docking site on the protease. These potent inhibitors were converted into affinity labeling reagents that contain a chemicaiiy reactive bromoacetyi or photoreactive benzophenone for covalent attachment to the protein target and a biotin moiety to allow isolation and detection of the labeled protein. Both types of chemical probes interacted with the two presenilin subunits but at distinct locations, which suggests that both the active site and the docking site of y-secretase lie at the interface between these subunits.

One of the two m or steps in the application of avidin-biotin technology is to incorporate the biotin moiety into the experimental system (6). This is usually accomplished by covalendy attaching biotin to a biologically active binder. The binder can be an antigen, a primary monoclonal or polyclonal antibody, a complement, interleukin-2, or any other component of the immune system. 

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