MICROPLATE READERS
the determined Km
. Tis is essential to
ensure that the limiting factor is the activity of the enzyme itself and not the amount of available substrate, since its amount is enough for the enzyme to run at full capacity. For the final enzyme kinetic, ideally enzyme and substrate concentrations as well as measurement time are chosen, so that approximately 10% of the total substrate is converted during a test run.
ADJUSTING MICROPLATE READER SETTINGS Measuring devices such as microplate readers offer further possibilities to define the best measurement window for a kinetic measurement. First, depending on the assay, selecting appropriate excitation and/or emission wavelengths is crucial for the generation of significant readout signals. Selecting these wavelengths based on peaks in the excitation/emission spectra of the used substrate will lead to the best results, while deviating from them can result in drops in signal strength. To a certain degree, this effect can be compensated by increasing the flash number of the excitation light source in fluorescence- based measurements or increasing the signal acquisition time window for assays which produce a long-lived emission signal, such as luminescence or time-resolved fluorescence measurements.
As most enzyme kinetics are based on the detection of fluorescent signals, one of the most important factors for setting the optimal assay window is an appropriate fluorescence gain value, which is the factor by which the incoming fluorescent signal is amplified. Typically, high gain values provide a large amplification and are hence suitable for dim signals. Very bright signals instead need a lower gain as less signal amplification is required (Fig. 2). Inappropriate fluorescence gain values negatively affect data quality, assay window and sensitivity. If bright samples are measured with a high fluorescence gain, this may result in the saturation of the detector and unusable data. On the contrary, if dim signals are detected with a low fluorescence gain, they may become indistinguishable from the background noise. To avoid these issues and provide the best possible dynamic range between the highest and the lowest measurement
Fig. 1. Rates plot of the NAD+-dependent ADP- ribosylation catalysed by a PARP-family enzyme
Fig. 2. Correlation between signal strength and fluorescence gain
values of your assay, fluorescence gain is typically adjusted on the sample with the expected highest signal output. However, kinetic assays pose a problem, as the light yield of the samples typically grows over time, increasing the chance of reaching saturation.
While all BMG Labtech microplate
readers can automatically determine the fluorescence gain, the enhanced dynamic range (EDR) technology on the Clariostar Plus and the Vantastar was specifically designed to offer the largest possible dynamic range with no manual intervention. Tis makes
gain adjustments superfluous as EDR ensures highly reliable results over a large dynamic range. Moreover, it simplifies the acquisition of kinetic experiments as it eliminates the risk of running into saturation.
Combining the above guidelines should give you a general idea for optimising kinetic enzyme studies
Martin Mangold is with BMG Labtech.
www.bmglabtech.com
www.scientistlive.com 27
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