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Labels, tags, and probes are relatively small modifying agents that can be used to label pro-teins, nucleic acids, and other molecules. These compounds often contain groups that provide sensitive detectability by virtue of some intrinsic chemical or atomic property such as fl uorescence, visible chromogenic character, radioactivity, or bioaffi nity toward another protein. Most probes can be designed to contain a reactive portion capable of coupling to the functional groups of biomolecules. After modifi cation of a protein via this reactive part, the probe becomes covalently attached, thus permanently tagging it with a unique detectable property. Subsequent interactions that the labeled protein is allowed to undergo can be followed through the tag ’ s visibility.

Labeling molecules by adding a radioactive component was one of the fi rst means of creating highly sensitive detection capabilities. Covalent modifi cation of activated aromatic rings with 125 I or 131 I easily can be done through tyrosine side chains in proteins or by the use of a phenolic-ring-containing modifi cation agent such as the Bolton–Hunter reagent (Chapter 12, Section 5). Other methods of introducing a radioactive isotope involve the intermediary use of metal-chelating modifi cation reagents such as diethylenetriamine pentaacetic acid (DTPA) (Chapter 10, Section 1). Heavy metal isotopes may be held in coordination complexes on a protein or other molecule and provide extreme sensitivity for in vivo diagnostic procedures involving the detec-tion of malignancies. Such complexes coupled to monoclonal antibodies also are being used in the treatment of cancer by their ability to cause cell death in proximity to the bound radiolabel.

Detection of probes or labels usually takes one of three general forms: spectrophotometric, radiosensitive detectors, or indirectly through another labeled substance. Spectral probes can be of two types, chromogenic or fl uorescent. Chromogenic labels typically are reserved for non-covalent staining of gross structural features within cells and tissues, as these are present at rela-tively high concentration. The sensitivity of visible wavelength dyes often is not good enough to provide detectability for low-concentration antigens or low-copy proteins. Even if a protein is covalently modifi ed with a chromogen, the number of associated dye molecules needed to detect it just through its absorbance properties could be prohibitively large to make it viable.

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fl uorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fl uorescently tagged antibodies can be used to probe cells and tis-sues for the presence of particular antigens, and then detected through the use of fl uorescence microscopy techniques. Since each probe has its own fl uorescence emission character, more

Fluorescent Probes

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