High-throughput applications assays

The combination of increased Pe and decreased %R allowed the permeation time to be lowered to 4 h, in comparison to the originally specified time of 15 h [547,550], a considerable improvement for high-throughput applications. The quality of the measurements of the low-permeability molecules did not substantially improve with sink conditions or the reduced assay times. 

G Wu, S Daniel-Issakani, K LaMarco, B Strulovici. Automated high throughput filtration assay application to polymerase inhibitor identification. Anal Biochem 245 226-230, 1997. 

Under confocal settings S/N ratios sufficient for high-throughput applications can be achieved with these fluorophores (Moger et al., 2006). Under standard optical settings, dynamic ranges far above 1 ns are required for robust screening assays. 

PT14 is comparatively cost efficient and thus affordable for high-throughput applications. We know of only one disadvantage of FLT as a readout for biochemical protease activity assays the requirement for a detection device equipped with a pulsed laser diode and TCSPC capabilities. According to our knowledge, such readers are currently offered by only two companies (Tecan and Edinburgh Instruments). 

For the assay of enzymes with products and reagents that have no absorption, fluorescence or luminescence in the ultraviolet or visible region, developments in analytical infrared spectroscopy can be used. In particular, mid-Fourier transform infrared (mFTIR) spectroscopy has been successfully applied to the determination of enzyme activities and kinetics, e.g. of /i-fructosidasc, phosphoglucose isomerase and polyphenol oxidase [90]. The method could very well be a tool that may also be applied to a variety of other enzyme classes. The potential of high-throughput applications, however, has yet to be demonstrated. 

AlphaScreen applications include assays for enzymes such as protein kinases and proteases, immunoassays such as cAMP detection, and protein-protein and protein-DNA interactions. A recent literature example is a comparison of AlphaS-creen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. In this comparison, the AlphaScreen system showed the highest sensitivity and the broadest dynamic range [149]. Another recent publication concerns a high-throughput binding assay for a TNF receptor [175]. 

Although a few studies have described medium-throughput microfluidic cell assays, the potential of this application has not yet been fully realized. Quake and coworkers [16] developed a device comprising hundreds of reaction chambers, a complex network of microchannels, and multiplexed valves. The utility of the device was illustrated by detecting a recombinant cytochrome c peroxidase (an enzyme used for chemiluminescent detection) expressed in E. coli. This device, which houses 256,725 nL chambers, demonstrates the potential for high-throughput cell assays with minimal reagent use. A similar platform was developed by Chin et al. [17] and contains 10,000 microwells (Fig. 3a). Rat neural stem... 

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