Dilution schemes

Figure 5.8 Dilution scheme for testing the reversibility of an enzyme inhibition The enzyme and inhibitor are pre-incubated at a concentration of enzyme equal to 100-fold that needed for activity assay, and at a concentration of inhibitor equal to 10-fold the IC50 value. The sample is then rapidly diluted 100-fold into an assay solution. The inhibitor concentration thus goes from 10-fold above die IC50 (corresponding to 91% inhibition) to 10-fold below the IC50 (corresponding to 9% inhibition).

The next question to be addressed is what the maximum concentration of inhibitor should be at the start of the 1.5-fold dilution series. Murphy suggests starting the dilution series at a concentration of inhibitor equal to 30[ ]T (or more correctly 30 times ones best estimate of total enzyme concentration). Simulations suggest that this dilution scheme will provide adequate data points within region B for inhibitors with potencies ranging from A fpp/[ ]T = 0.01 to 10. 

Use of Morrison s quadratic equation, together with Murphy s recommended dilution scheme, will allow accurate estimates of Kfpjpj as low as 100-fold below the total enzyme concentration. Based on Murphy s simulations, the most accurate determination of Kfpp is obtained for inhibitor titrations performed at / T = 10A I, (Murphy, 2004). 

The same dose-response dilution schemes as described for Levels 2 and 3 are used for these assays, and the IC50 is determined. The compounds having the widest spectrum of nanomolar (nM) antitumor potency and an SI of 3 or greater for a panel of normal cells are considered to be "Hits" in the screen and to merit further evaluation and testing. 

This further incubation should be sufficiently long enough to observe measurable metabolite formation. There is some debate as to the most ideal dilution scheme that should be followed, but the general consensus is that a higher dilution (e.g., >10-fold) reduces the influence of competitive inhibition. Also the concentration of probe substrate should ideally be at least 5 Km, the purpose being that the high probe concentration together with the dilution step minimizes competitive inhibition of... 

The basic problem of direct fluorination involves both kinetics and thermodynamics. The rate of the reaction must be slowed down so that the energy liberated from the reaction may be absorbed or carried away. The most significant and crucial innovations in the evolution of the direct fluorination process recently developed by Lagow and Margrave (6) have been kinetic considerations. Their technique has been named the La-Mar direct fluorination process (7). Most of the kinetic considerations involve fluorine dilution schemes and probability considerations. 

Prepare a series of concentrations down to 0.1 ppm following the dilution scheme in Figure Gl.5.2. 

Prepare standards using the following dilution scheme ... 

Dilution scheme. Whenever possible, a sample requiring dilution to yield a report-able result should be reanalyzed using the same dilution factor as the original analysis. Samples should not be diluted to levels <3x the LLOQ. 

Dilution of the prealiquoted compound samples with assay buffer is performed just in time before the screen. A 384-well parallel pipettor on the HTS system adds assay buffer to the compound aliquots in the wells, mixes the diluted compound solutions in the wells, and transfers the amount of compound solution needed for the assay from the sample plate to one or multiple assay plates, which are processed in one screening run on the HTS system. This procedure minimizes degradation of unstable compounds caused by storage of compound collections in aqueous solution over days or even weeks. In addition, it allows very flexible dilution schemes in the assay plates without using intermediate dilution plates. 

The earliest examples of transition metal carbenoid C-H insertion reactions were carried out with ethyl diazoacetate 13 (EDA) and simple alkanes acting as the reaction solvent. In 1974, Scott and DeCicco reported that CuS04 and CuCl were capable of decomposing EDA in the presence of cyclohexane (14) to generate C-H insertion product 15 [13]. Although these results, along with control experiments, provided proof that the copper catalyst was necessary for the transformation to take place, the maximum yield of the product was only 24%, and dimeric byproducts of the diazo compound dominated, even at high dilution (Scheme 3). 

In the case of commercial kits, comparison of kit and reagent lots should be addressed early on and, due to cost, the scope and goal of the transfer and development should be clearly defined. For example, use of additional or modified controls, modified standard curves, and sample treatment options (dilution scheme, assessment of minimal required dilution depending on matrix concentration) should be included. 

Sample Preparation (Treatment) No treatment, extraction, leaching, slurry preparation, decomposition, wet (acid/alkaline) digestion, dry ashing, fusion, combustion (bomb/tube), ignition, pyrolysis, avoidance of losses and contamination, complete decomposition/dissolution, dilution scheme... 

The authors conclude that if heteroscedaslicity is ignored, confidence intervals will be too narrow at the high end and too wide at the low end of the ICP calibration curve. The magnitude of these effects will depend on the particular dilution scheme used to make the calibration standard solutions. How does the dilution scheme for standards affect the results of least-squares analysis on ICP calibration curves ... 

Figure 1 shows diagrammatically the dilution scheme. The upper part of Fig. 1 illustrates the typical numbering and lettering associated with microtiter plates. Thus, columns are labeled rC12 and rows are labeled A CH. This nomenclature is used henceforth to identify locations on the plates. 

Several syntheses of carotenoids isotopically labelled with deuterium have been reported [65-68]. The total synthesis of spheroidenes (97) specifically labelled with deuterium in the central part is based on the synthetic scheme discussed above for the C-labelled spheroidenes [68]. When deuterium-enriched compounds are used, a few modifications are necessary to avoid scrambling and isotope dilution (Scheme 28). 

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