Biotin-fluorophores

Although biotin-fluorophores have been described in the detection of biomolecules, namely proteins, peptides, or DNA, most of these loose part of their fluorescence when binding to the tetrameric proteins avidin and streptavidin. 

Gruber HJ, Marek M, Schindler H, Kaiser K. Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin. Bioconj. Chem. 1997 8 552-559. 

The total synthesis of a novel hybrid lipid Pea-PIP2 (51), possessing a phosphatidyl ethanolamine (PE) headgroup at the 1-position and a phosphatidyl inositol 4,5-bisphosphate [PtdIns(4,5)P2] headgroup at the 4-position has been elaborated. Reporter groups (biotin, fluorophores, spin label) were... 

Activity-based probes (ABPs) are tagged covalent and irreversible enzyme inhibitors. Formation of a stable covalent bond ensures that the inhibitor will remain attached to the polypeptide after protein denaturation, after which the tag (radio-isotope, biotin, fluorophore) allows visualization and/or identification of the thus modified enzyme or enzyme family. The first proteasome ABP described comprised a tritium-labeled lactacystin analog [6,7]. Proteasome bands are visualized on one-dimensional SDS-PAGE (sodium docecylsulfate-polyacrylamide gel electrophoresis) gel in an autoradiogram after treatment with a radiolabeled... 

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] ... 

A common application for (strept)avidin-biotin chemistry is in immunoassays. The specificity of antibody molecules provides the targeting capability to recognize and bind particular antigen molecules. If there are biotin labels on the antibody, it creates multiple sites for the binding of (strept)avidin. If (strept)avidin is in turn labeled with an enzyme, fluorophore, etc., then a very sensitive antigen detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through its multiple biotinylation sites is the key to dramatic increases in assay sensitivity over that obtained through the use of antibodies directly labeled with a detectable tag. 

Jackson ImmunoResearch Laboratories Inc (http //www.jacksonimmuno.com/) sells all the components to develop in-house such staining. Suitable but rather expensive kits exploiting monovalent Fab fragments bearing fluorophore or biotin label have also been developed commercially, such as Animal Research Kit (ARK) by Dako (http //www.dakousa.com/) and Zenon Labeling Kits by Molecular Probes (http //probes.invitrogen.com/products/zenon/). 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at + 4°C with a correspondingly diluted primary antibody labeled with a fluorophore or biotin. Wash sections in PBS for 2 x 3 min. 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

Visualization of biotin biotin can be visualized using (strept)avidin conjugated with a fluorophore of with an enzyme label. 

When using biotin-labeled secondary antibody, you have first to visualize biotin with a fluorophore-labeled (strept)avidin employing ABC technique (see Sect. 6.2.1), before proceeding to counterstaining (step 7). 

Another approach to this persistent problem relies on haptenylation of primary antibodies. Hapten (e.g., biotin, digoxigenin or any fluorophore) can be covalently bound to the antibody via A-hydroxysuccinimide esters (NHS-ES) (see Sect. 2.1), or conjugated employing monovalent IgG Fc-specific Fab fragments (see Sect. 2.2). Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (Fig. 8.5). Fluorophore-labeled primary antibodies can be directly visualized in a fluorescent microscope. 

Notes The label may also be biotin or any fluorophore. For antibody biotinylation, we use biotin-SP-conjugated AffiniPure Fab Fragment Goat Antimouse IgG (H+L) (Jackson ImmunoResearch Labs, Code Number 115-067-003). 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

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