Probes labeling with protein

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

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 fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

Many of the methods developed to study protein interactions use the bait/prey model to detect interacting partners (Phizicky and Fields, 1995 Archakov et al., 2003 Piehler, 2005). The bait protein is a purified protein (often recombinant) that is used to lure and capture a putative interacting protein or biomolecule. The bait protein may be immobilized to a solid phase for affinity separations or be used in solution. It also may be fusion tagged (i.e., GST or 6X His) or labeled with a detectable molecule, such as a fluorescent probe. It often is the case... 

The results obtained show that the dipole-relaxational motions in protein molecules are really very retarded as compared to such motions in the environment of aromatic molecules dissolved in liquid solvents (where they occur on a time scale of tens of picoseconds).(82) Dipole-relaxational motions on the nanosecond time scale have been observed for a variety of proteins. For example, such motions were recorded for apohemoglobin and bovine serum albumin0 04 105) labeled with the fluorescent probe 2,6-TNS. 

This labeling has been used with success in yeast where 6 of the 17 known DUBs were labeled with UbVS [115]. Incomplete labeling likely results from DUBs that do not act on mono-ubiquitin or where the UbVS could not access the active site. The labeling has also been used with great success in mammalian cell lysates to identify novel ubiquitin DUBs [41]. A novel deneddylating enzyme and a novel DUB that acts on autophagy-related UbL proteins have also been identified using vinyl sulfone labeled probes [26, 37, 116]. 

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