Functional assays methods

Tang WM, Kang JS, Wu XY et al (2001) Development and evaluation of high throughput functional assay methods for hERG potassium channel. J Biomol Screen 6(5) 325-331... 

A useful method of weighting is through the use of an iterative reweighted least squares algorithm. The first step in this process is to fit the data to an unweighted model. Table 11.7 shows a set of responses to a range of concentrations of an agonist in a functional assay. The data is fit to a three-parameter model of the form... 

Free drug concentration description of, 36-37 measurement of, in receptor compartment, 39 Frovatriptan, 163f Full agonism, 200-202 Full agonists affinity of, 261 description of, 27—30 dose-response curves for, 90, 200-202 Furchgott method for affinity measurements, 261 potency ratios for, 202—204, 219—220 Functional assays... 

In principle, the use of addressable, pooled GST fusion proteins could be used to identify proteins associated with any biochemical activity, assuming that the fusion protein is soluble, folded and functional. The method has the additional advantage that, once the GST fusion clones are constructed, it is a rapid technique. The authors state that only two weeks are required to purify the 64 pools and the assays can be accomplished in a day (Martzen et al., 1999). In addition, the method is sensitive because only 96 recombinant proteins are assayed at one time in contrast to the use of cell lysates where thousands of proteins are present. This leads to a much higher concentration of each protein, which greatly facilitates detection of a biochemical activity (Martzen et al., 1999). 

Arand, M., Cronin, A., Adamska, M. and Oesch, F. (2005) Epoxide hydrolases structure, function, mechanism, and assay, Methods in Enzymology, 400, 569-588. 

Coller, J., and Wickens, M. (2007). Tethered function assays An adaptable approach to study RNA regulatory proteins. Methods Enzymol. 429, 299-321. 

Wilson, S.D., Munson, A.E. and Meade, B.J., Assessment of the functional integrity of the humoral immune response the plaque-forming cell assay and the enzyme-linked immunosorbent assay, Methods, 19, 3, 1999. 

Malabsorption Syndrome, with Special Reference to the Effects of Wheat Gluten (Frazer), 5, 69 Mellituria, Nonglucose (Sidbury), 4, 29 Microbiological Assay Methods for Vitamins (Baker and Sobotka), 5, 173 Organic Acids in Blood and Urine (Nordmann and Nordmann), 4, 53 Paper Electrophoresis Principles and Techniques (Peeters), 2, 1 Paper Electrophoresis of Proteins and Protein-Bound Substances in Clinical Investigations (Owen), I, 238 Parathyroid Function and Hyperparathyroidism, Biochemical Aspects of (Nordin), 4, 275... 

Significant advances have been made in the preparation of discrete macromolecules that include both coenzyme function and a defined polypeptide or protein architecture. Preliminary, but promising, functional studies have been carried out and assay methods developed. While in many cases rather modest effects have been observed, what is significant is that the methodology exists to prepare, characterize, and study defined macromolecular constructs. With new information becoming available on co enzyme-dependent protein catalysts from structural biology and mechanistic enzymology, it should be possible to more fully exploit the remarkable breadth of coenzyme reactivity in tailored synthetic systems. 

Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron), (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein, (b) Analysis of the same natural extract spiked with digoxin using the label-free MS assay method as shown in Fig. 5.15.

Aminoglycosides remain clinically important antibiotics. NMR provided the initial breakthrough in structural understanding of aminoglycoside action on the ribosome, and it remains a powerful tool for the biophysical characterization of drug-RNA interaction. The combined use of NMR, X-ray crystallography, thermodynamic and functional assays, and computational methods is needed to drive forward the development of new aminoglycosides with improved clinical properties. The rich data described above, combined with the application of new synthetic methods, bode well for the future. 

For this functional assay we use the Beta-Glo assay (Promega), a luminescent assay able to quantify Beta-Gal expression. Other methods allowing a sensitive measurement of the reporter could be adopted (see Note 11). 

Thus, cell surface markers remain a useful tool and have widespread use, as evidenced by the stem cell literature. Limitations, explained in the bone marrow transplantation literature, highlight the need for the development of new methods for identifying stem cells in the emerging field of cell therapy for cardiac diseases. Indeed, functional assays will likely play an important role in stem cell selection and classification in the future. 

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