Proteins labelling with fluorophores

The energetics of protein association can be studied by a variety of experimental techniques,17 each of which permits measurements of equilibrium or kinetic values in a certain range. Widely used techniques include isothermal titration calorimetry, surface plasmon resonance measurement, stopped flow kinetics, optical spectroscopy, MS, and analytical ultracentrifugation. The techniques differ in their requirements (e.g., amount of protein, labeling with fluorophores, attachment to sensor surfaces, and the environment provided by the experimental set up) and therefore in their applicability to individual cases. Different techniques can also give quite different values for what might be expected to be the same quantity. For example, association rates measured by surface plasmon resonance, with one protein immobilized on a surface, are usually different from those measured for the two proteins in solution and under otherwise similar conditions. 

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

Labeled proteins are used in enzymology especially as substrates for determining the activity of proteolytic enzymes, for studying structural properties of enzyme molecules (7.2), and for some analytical purposes. Here, attention is paid to the determination of proteolytic activities when proteins labeled with radionuclides, chromophores and fluorophores are used as substrates. 

Urwin, V. E. and Jackson, R. (1993) Two-dimensional polyacrylamide gel electrophoresis of proteins labeled with the fluorophore monobromobimane prior to the firstdimensional isoelectric focusing imaging of the fluorescent protein spot patterns using a cooled charge-coupled device. Anal. Biochem., 209, 57-62. 

While dansyl chloiide today seems like a common fluorophore, its Introduction by Professor Weber represented a fundamental change in the paradigm of fluorescence spectroscopy. One of Professor Weber s main contributions was the introduction of molecular considerations Into fluorescence spectrosetpy. The dansyl group is solvent sensitive, and one is thus forced to consider its interactions with its local environment. Professor Wfeber recognized that proteins could be labeled with fluorophores, which in turn reveal information about the proteins and their interactions with other molecules. The probes which the Professor developed are still in widespread use, including dansyl chloride, ANS, TNS, and Prodan derivatives. 

Triton X-100). Alternatively, cells can be fixed and permeablized with organic solvents such as methanol and acetone. The type of fixative depends on the cellular components that need to be detected and the employed antibodies. Cellular components are then detected either directly by means of dyes (e.g., fluorescent dyes that bind to and stain DNA) or antibodies that are labeled with fluorophores. Alternatively, an indirect detection can be performed by means of primary antibody that specifically binds to a protein of interest and a secondary antibody that recognizes the primary antibody and is labeled for detection (usually with a fluorophore). Finally, samples are analyzed by means of fluorescence microscopy. 

In conclusion, silver particles or colloids, when bound to a protein heavily labeled with fluorophores, can provide significantly higher intensities due to a decrease in the extent of self-quenching. 

Recently, Beatty and Tirrell [201] relied on the simultaneous or sequential addition of two reactive Met analogs, Aha and Hpg, to enable the fluorescent tagging of two protein populations within cells. The first demonstration of two-dye labeling of metabolically tagged cells was described in 2007 by Chang and co-workers [202], who used flow cytometry to show that cells treated with two reactive sugars could be labeled with distinct fluorophores. 

FRET is an extremely useful phenomenon when it comes to the analysis of molecular conformations and interactions. F or the analysis of interactions, in which two separate molecules are labeled with an appropriate pair of fluorophores, an interaction can be shown by observing FRET. Further, FRET can be used as a type of spectroscopic ruler to measure the closeness of interactions. Proteins, lipids, enzymes, DNA, and RNA can all be labeled and interactions documented. This general method can be applied not only to questions of cellular function like kinase dynamics [3] but also to disease pathways, for example, the APP-PS1 interaction that is important in Alzheimer s disease (AD) [4], Alternatively, two parts of a molecule of interest can be labeled with a donor and acceptor fluorophore. Using this technique, changes in protein conformation and differences between isoforms of proteins can be measured, as well as protein cleavage. 

A solution of a pure fluorophore may reasonably be expected to display a single exponential decay time. The emission from fluorophore-protein conjugates, on the other hand, may be best characterized by two or three exponential decay times (Table 14.2). In labeling proteins with fluorophores, a heterogeneity of labeled sites results in fluorophore populations that have different environments, and hence different lifetimes. The lifetime distribution of a fluorophore-protein conjugate in bulk solution may vary further when immobilized on a solid support (Table 14.2). 

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