Fluorescent beads

Fig. 1. Various schematics of bead display for molecular assemblies on beads. The Py subunits of the G protein (circles labeled with [i and y) are fused with either FLAG or hexahistidine tag, which recognizes the biotinylated M2 anti-FLAG antibodies on streptavidin-coated beads or chelated nickel on the dextran-treated beads. A socket and plug connecter is utilized to depict the very high-affinity interaction of the epitope tag. This modular setup allows for either a subunit (for capturing FPR-GFP) or as subunit (for capturing / 2AR-GFP) to be coupled with the fly subunit to form the complete G protein coating the bead. Fluorescent components such as GFP or ligand are indicated in green. See text for details.

The on-resin screening of one-bead-one-peptide libraries for enzyme inhibitors is possible when a quenched fluorogenic substrate, converted into a fluorescent molecule by the enzyme, is present on each bead along with the library peptide. l After incubation of the library beads with the enzyme, most beads fluoresce, since the substrate is cleaved and the fluorescent part of the molecule remains attached to the bead. Only beads containing peptides that inhibit the enzyme do not fluoresce, since the substrates bound to these beads are not converted. 

Surface-modified nanobeads, beads where the chelates have shorter distance to the respective acceptor-labeled reactant, are also used to create proximity in TR-FRET assays such as cAMP immtmoassay and assays of kinase activities by the Molecular Devices IMAP system. The kinase assays apply fluorescent peptides as substrates, which after phosphorylation are collected to beads by complex formation between phosphor and the trivalent metalhc ions on the beads. Fluorescent terbium chelates, substituted with a phosphor group, provide thereafter the energy-donating groups to create sensitized signal on bead surfaces [37]. 

Polymer—Cp—MCl complexes have been formed with the Cp-group covalendy bound to a polystyrene bead. The metal complex is uniformly distributed throughout the bead, as shown by electron microprobe x-ray fluorescence. Olefin hydrogenation catalysts were then prepared by reduction with butyl hthium (262). 

Sheetz, M.P. Spudich, J.A. (1983). Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature 303, 31-35. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Nath S, Maitra U (2006) A simple and general strategy for the design of fluorescent cation sensor beads. Org Lett 8 3239-3242... 

Hasemeier B, Christgen M, Kreipe H, et al. Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology. BMC Biotechnology 2008 8 90, doi 10.1186/1472-6750-8-90. 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

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