Marker substrate concentration

The CYP marker substrate concentration should be equal to or less than the Km for the reaction. 

For IC5o determinations, the substrate concentration should be close to the Am for the marker reaction. As discussed previously, this choice of substrate concentration allows an estimate of the A) value because IC50 = 2A) for competitive inhibition and IC50 = A, for noncompetitive inhibition. For A) determinations, a common substrate concentration scheme is Am/3, Am, 3Am, 6Am, and 10Am. Assuming that the Km for the reaction has been accurately determined, this range of substrate concentrations will provide an adequate spread of data on an Eadie-Hofstee plot to readily observe the mechanism of direct inhibition. For some substrates, solubility can become limiting at concentrations >2Am. In such cases, it becomes necessary to choose alternate concentrations so that no fewer than five concentrations are used in a A, determination. The choice of substrate... 

Each drug (at least in five concentration which covers two orders of magnitude) is incubated with human liver microsomes in the presence of the marker substrate. Reactions are initiated with addition of NADPH... 

The advantages of SPR experiments are that only small amounts of sample are required,72 often hundreds of microliters of solutions with nanomolar to micromolar concentration of reactants and the substrate attached to the surface can oftentimes be reused after washing in buffer. The fact that changes in the refractive index values are measured avoids the need to use absorption or fluorescence markers to follow the binding kinetics. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

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