Assay media

PURPOSE AND RATIONALE Solubility assays are gaining growing attention in drug discovery, because many pharmaceutically active compounds can be adjusted to in vivo testing merely with co-solvents. Furthermore, in vitro assays may also lead to false results, simply for precipitation of a compound in the assay media. Solubility assays vary in one main point they are either performed from solids or stock solutions. A nomenclature has been established in the literature which tries to distinguish between these methods. Determinations from stock solutions are often called kinetic solubility whereas thermodynamic solubility stands for solubility of solids (Kerns). Thermodynamic solubility takes the crystal lattice forces into account. Batch to batch variations, polymorphism... 

Figure 9.41 Chromatograms of enzyme assay media. (A) Elution profile of the assay medium of glutamine synthetase. Alder root nodule enzyme plus assay mixture was incubated for IS minutes. (B) Elution profile of the assay medium of glutamate synthetase. Rice leaves enzyme plus assay mixture was incubated for 15 minutes. (From Martin et al., 1982.)...

Figure 9.45 Chromatography of enzyme assay media. Peaks 1, aspartate 2, glutamate 3, asparagine 4, glutamine 5, Tris-HCl buffer. Elution profile of the assay medium incubated for (A) zero time and (fl) 30 minutes. (From Unnithan et al., 1984.)...

The 2 -azido group of cytarazid renders the nucleoside more resistant to deamination to the 2 -azidouridine derivative (153) by deoxycytidine deaminase, but was also observed to reduce substrate affinity for the deoxycytidine kinase necessary for anabolic phosphorylation to the active cytarazid 5 -triphosphate [180]. Conversely, cytarazid was a more potent inhibitor of the target DNA polymerases a and K = 0.6 and 0.7 //M, respectively) than the parent ara-C (.A = 10 and 17 fiM, respectively), and the dissimilar spectrum of antitumour activity exhibited by the two compounds was attributed to differences in stability, metabolic activation and inhibitory potency [180, 181]. Interestingly, the instability of cytarazid to thiols present in the assay media, was commented on but not pursued [180]. In view of previous discussions concerning the bioreduction of AZT... 

Inert carrier proteins, such as bovine serum albumin, are commonly added to the assay media at high concentrations for screening of isolated targets in order to saturate the potential protein binding surfaces and reduce the loss of target proteins due to adsorption onto the surfaces of the containers, such as plates and vials. The added benefit of inert proteins is that they can bind to lipophilic compounds and increase the solubility of insoluble compounds in the assay buffer. The presence and absence of serum protein can cause difference in solubility and cause a discrepancies between the two assays. 

Several strategies have been developed to overcome solubility issues in assay media (Table 4). These are discussed below. 

ASTM, Evaluation of Air Assay Media by the Monodisperse DOP Smoke... 

Assay media that had been characterized as ideal for the growth of that particular strain and that could also be obtained lacking a specific nutrient were commercially available. 

Assay media prescribed composition used for assaying vitamins, amino acids and antibiotics. 

Kokko L, Lovgren T, Soukka T (2007) Europium(ni)-chelates embedded in nanoparticles are protected from interfering compounds present in assay media. Anal Chim Acta 585 17-23... 

In Vitro STxB Transport Assay Media and buffers... 

Media Assay Disc Assay Media Assay Disc Assay Media Assay Disc Assay... 

Superoxide radical (Oj "), generated in a hypoxanthin xandiine oxidase system was also scavenged readily and conqiletely in assay media containing 200 ppm of extracts of borage and evening primrose. Almost all fractions of exfracts also effectively scavenged 02 as evidenced by die absorption of the ink-bue color of die system at 560 ppm. 

Media incubated above 55 C. are virtually free from microbial contamination. Assay media need not therefore be heat-sterilized, thus obviating the risk of destroying thermolabile forms of the vitamin. If the co-factor, i.e., enzymatic forms of vitamin Bi2 resemble those of the other B vitamins, they are likely to contain such thermolabile constit-... 

A substantial literature bears witness to the importance of investigating the effects of h amic substances on enzyme changes in plant tissues. In such studies it is usual to distinguish between an effect on enzyme development (synthesis), and an effect on enzyme activity yev se which is measured by adding the humic substances to the enzyme assay media. In this section, only protein synthesis generally and enzyme synthesis in particular, are considered because they relate directly to translation and/or transcription involving m-RNA. 

Photobleaching. Ten ml aliquots of a 20 pg Chi. ml suspension of thylakoids in assay media (0.33 M sorbitol, 3 mM MgCl, 10 mM NaCl, 30 mM phosphate buffer pH 6.5) were pr ared just prior to irradiation. These samples were dispensed into 20 ml test tubes and placed in a thermostated water bath at 2°C. Irradiation was provided by a white light projector lamp delivering 10.2 m Mole, m . s PAR. 

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