Alexa Fluor probes

Fig. 1 Glycan array analysis of codakine as measured by fluorescence intensity (glycan array v3.0 at Consortium for Functional Glycomics). Purified codakine from white clams was purified with Alexa Fluor 488 Protein Labeling Kit (Molecular Probes , Invitrogen) and tested on glycan array v3.0 at Consortium for Functional Glycomics.

Dramatic advances in modem fluorophore technology have been achieved with the introduction of Alexa Fluor dyes by Molecular Probes (Alexa Fluor is a registered trademark of Molecular Probes). Alexa Fluor dyes are available in a broad range of fluorescence excitation and emission wavelength maxima, ranging from the ultraviolet and deep blue to the near-infrared regions. Because of the large... 

Anti-Aurora-A (see Note 1) monoclonal antibody (610939, BD) and Alexa Fluor 488 Goat Anti-Mouse (A-11001, Molecular Probes). 

The target molecule can be directly labeled with a fluorescent dye or it can be indirectly labeled via biotinylation and successive incubation with a streptaviclin, R-phycoerythrin conjugate. We recommend to biotinylate the target protein since it can be used both for MACS and for FACS then. For direct fluorescence labeling of the target molecule, NHS-coupled fluorescence dyes, e. g., Alexa Fluor 488 from Molecular Probes, can be used, following the procedure described in protocol 2. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Alexa Fluor 546-labeled Transferrin conjugate from Molecular Probes, Eugene, OR to stain endosomes. 

Fig. 20.2. Photo-switchable probes constructed from activator-reporter pairs, (a) Spectrally distinct reporters exhibit photo-switching behavior. The lower panel shows the fluorescence time traces of Cy5, Cy5.5, and Cy7 when paired with a Cy3 dye as the activator. The upper panel shows the green laser pulses used to activate the reporters. The red laser was continuously on, serving to excite fluorescence from the reporters and to switch them off to the dark state, (b) The same reporter can be activated by spectrally distinct activators. The lower panel shows the fluorescence time traces of Cy5 paired with different activators, Alexa Fluor 405 (A405), Cy2, and Cy3. The upper panel shows the violet (405nm, magenta line), blue (457nm, blue line), and green (532 nm, green line) activation pulses...

Cascade Blue , Alexa Fluor , Texas Red are registered trademarksofMolecular Probes, Inc. 

Dextran conjugates (dextran, Alexa fluor 488, -594, cascade blue 10,000 MW, anionic, fixable) (Molecular Probes, Invitrogen). 

Secondary antibodies Alexa FluorTM488-conjugated, Alexa Fluor 568-conjugated (Molecular Probes, Carlsbad, CA), and cyanin-5-conjugated (Jackson ImmunoResearch Laboratories, West Grove, PA) donkey anti-mouse, anti-rat, anti-rabbit, and anti-goat antibodies. 

Resuspend isolated A. phagocytophilum in 1 mL PBS containing 7.5 pg/mL Alexa Fluor 488 (Invitrogen-Molecular Probes). Incubate for 15 min at room temperature. Wash twice in 1 mL PBS to remove unbound AlexaFluor 488. Resuspend bacteria in RPMI/HEPES at desired concentration to add a volume of 250 pL of suspended bacteria to each well. 

Primary antibody (1° ab) mouse anti-FLAG Ml antibody (Sigma) Secondary antibody goat anti-mouse IgGl, Alexa Fluor 488 conjugate (Molecular Probes)... 

Combination of europium- and terbium-labeled oligonucleotide probes with complementary probes labeled with quenchers has been used in competitive hybridization assay as to screen celiac disease-related alleles [23] and cystic fibrosis risk alleles [42]. Doubly labeled beacon-type probes containing europium chelate as donor and either Alexa 647 or Alexa Fluor 700 as acceptors has been utilized by measuring both the wavelength and decay change upon hybridization [43]. 

Figure 3.22 Schematic of the simple confocal microscope that produced much of the data not otherwise referenced in this text. Specific examples of components are given for a configuration to measure diffusion spFRET or FCS (autocorrelation or cross-correlation) for the dye pair Alexa Fluor 488 and Alexa Fluor 594 (Moelcular Probes Inc., USA).These examples represent, in most cases, an arbitrary choice of supplier and are provided only to enable the user to see the specifications of the parts used. (Figure was kindly produced using Zemax (Zemax Development Corporation) by Kurt Baldwin at Avacta Ltd www.avacta.com).

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