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Tutorials : May 1, 2008 ( )
Boosting Microplate Reader Functionality
The Addition of Absorbance-Measurement Capabilities Enhances Filter-Based Systems
An ever-increasing pool of potential therapeutic targets is now available, largely as a result of the successful deciphering of the human genome five years ago. Hence, researchers in life science and pharmaceutical companies have set out to better understand the basic mechanisms underlying acute and chronic diseases such as diabetes, Alzheimer’s, and chronic pain.
Microplate readers are central to this work. Their utilization in basic research and drug development has benefitted tremendously as a result of automation and miniaturization of microplates. The implementation of 384- and 1,536-well plates along with new technologies have definitely streamlined the use of assays for particular targets. As a result, kinase profiling, protein-protein binding, the study of GPCRs, and cell-based assays are increasingly subject to refinement and thus becoming more and more sophisticated.
Filters offer many advantages over monochromators including more light transmission and excellent blocking of undesired wavelengths, higher sensitivity, precise control over transmitted peak shape, fast switching to other wavelengths when more than one filter pair is employed, and lower cost.
BMG Labtech’s FLUOstar Optima and POLARstar Optima readers are capable of measuring a range of assays and offering simultaneous dual emission for FRET, BRET, and fluorescence polarization measurements. Full-spectrum absorbance measurements, however, are not possible.
To address that shortcoming, BMG has incorporated Tandem Technology into its Omega microplate-reader series (Figure 1). Tandem Technology combines an ultrafast UV/Vis full-spectrum absorbance spectrometer built into a microplate reader and high-performance optical filters.
The ultrafast UV/Vis absorbance spectrometer in the Omega series features efficient optical grating and a solid-state array detector that allows the measurement of light intensity throughout the UV and visible parts of the spectrum.
With this instrument, full-spectral absorbance can be captured at high speeds from 220–850 nm at a resolution of 1 nm. Unlike a monochromator, the spectrometer allows users to capture the entire UV/Vis spectrum of a sample in approximately one second per well—no scanning needed. If users choose to reduce data-set size, up to eight discrete wavelengths can be selected and data will be collected simultaneously.
Further, microplate data analysis and reduction software (MARS) automatically creates a standard curve and calculates the concentration for unknown samples. If the option “path length correction” is used, the measured data is normalized to a path length of 1 cm, thereby allowing a comparison to be made between absolute data obtained from a microplate reader and data obtained from a cuvette-based spectrometer.
Validation of Technology
The validation of Tandem Technology was performed using the FLUOstar/ POLARstar Omega reader. The Bradford Assay (protein binding) and the determination of DNA content (A260/A280 ratio) were used to validate the absorbance capacity of the UV/Vis spectrometer.
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