Spectroscopic assays fluorescence

In this paper we review the fluorescence and spectroscopic assays commonly used in our lab to study neutrophil activation. The advantages of these assays are threefold. (1) These assays are very sensitive. (2) These are kinetic assays which can be measured with a time resolution of 1 s or less. This is important since many of these responses occur within seconds of stimulation and are... 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Competition-in-solution assays can quantify the effect of library components on the interaction between components of a protein complex. These assays include traditional biochemical assays that measure interactions through antibody- or tag-mediated pull-down or label displacement assays, along with biophysical methods that measure changes in the property of a component upon binding to its partner protein, such as fluorescence spectroscopic assays and SPR. It is essential for assay development that the requirements (affinity and binding site) for assembly of the interaction partners be at least partially defined such that a tractable model system for the target interaction can be developed, as was the case for MDM2/p53, IL-2/IL2-aR and Bcl/Bak [138-140],... 

K.-Z.Liu,R.A.Shaw,T.C. Dembinski,G. J. Reid,S.L.Ying,andH.H.Mantsch, Gomparison of Infrared Spectroscopic and Fluorescence Depolarization Assays for Fetal Lung Maturity, Am.. Obstet. Gynecol., 183(1), 181 (2000). 

Spectroscopic methods such as uv and fluorescence have rehed on the polyene chromophore of vitamin A as a basis for analysis. Indirectly, the classical Carr-Price colorimetric test also exploits this feature and measures the amount of a transient blue complex at 620 nm which is formed when vitamin A is dehydrated in the presence of Lewis acids. For uv measurements of retinol, retinyl acetate, and retinyl palmitate, analysis is done at 325 nm. More sensitive measurements can be obtained by fluorescence. Excitation is done at 325 nm and emission at 470 nm. Although useful, all of these methods suffer from the fact that the method is not specific and any compound which has spectral characteristics similar to vitamin A will assay like the vitamin... 

Selected entries from Methods in Enzymology [vol, page(s)] Design, 178, 551 immunoassay, 178, 542 production, 178, 531 purification, 178, 543 substrates and enzymatic assay, 178, 544 derivatization with spectroscopic probe, 178, 567 ester cleavage assays, 178, 565 fluorescence quenching binding assay, 178,... 

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

Majority of HTS is performed in microplates most frequently used formats contain 96, 384, and 1,536 wells per plate (Fig. 2). Consequently, HTS assays require detection approaches that could be easily executed in a plate format. Although the number of novel detection approaches adapted in HTS is constantly growing, assays based on spectrophotometric properties are by far the most prevalent, reflecting their technical and technological simplicity and low cost. Absorbance, fluorescence, luminescence, and derivations thereof are common in HTS assays. Broad selection of relatively inexpensive monochromator-, optical filter-, or diode array-based plate readers and suitable generic plates provides a foundation to spectroscopic HTS assays. 

Fig. 2.3. (a) Fluorescence spectroscopic changes observed in a proteolytic enzyme assay using PPE-SO3 (3) and K-pNA. Initial addition of KpNA quenches fluorescence, and then addition of peptidase gives rise to fluorescence recovery. Solid line Initial fluorescence, [PPE-SO3] = 1.0 pM, phosphate buffer solution, pH 7.1 Dotted line fluorescence after addition of 167 nM K-pNA. Fluorescence intensity as a function of time (5-200 min) after addition of porcine intestinal peptidase (3.3 pg mL ). (b) Mechanism of the turn-on CPE-based sensor... 

---