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Adobe acrobat dc calculations6/1/2023 ![]() In a luciferase reaction, light is emitted when luciferase encounters oxygen, ATP, and magnesium in a series of reactions. Luciferase comes from a number of sources such as fireflies. Luminescent assays emit light via a chemical reaction and often use luciferase. When working with light sensitive reagents, be sure to keep them covered to prevent photobleaching and ruining the experiment. In fluorescent-based assays, a fluorochrome is activated by a certain wavelength of light and in turn causes excitation of the fluorochrome, which emits light at a different wavelength. This compound binds to the proteins in the sample, and cause a shift in its absorbance. The Bradford assay is an example of an absorbance-based microplate reader assay, where protein samples are added to the plate with the “Bradford” reagent. Many types of microplate readers measure absorbance, which is defined as the logarithmic ratio of light falling upon an object to the light transmitted through an object. Concentration values can also be estimated by drawing a line from the absorbance value on the Y axis to the best fit line and then down to the X axis. Using the line of best fit, we can calculate the concentration values of experimental samples or controls in each well by plugging in the absorbance value for Y and then solving the equation for X. The coefficient of determination, a statistical measure of how well the line predicts actual data points, should be between 0.90-0.99, with 0.99 being considered the best value and signifies that the line fits the data perfectly. This can be easily done using a spreadsheet program. When the values have been plotted, the line of best fit can be calculated using a linear regression. Once the plate is read, use the average value of the blank samples to subtract the background from all samples including the standard curve.Īfter reading, the known concentration values for the standards are plotted against their respective measured values, absorbance in this case. Once the tray is loaded, parameters such as the mode, wavelength and well loading order are set in the software before the plate is read.Īfter the parameters are set, the plate is read, and the reader generates a read-out of values within the software. Remember to exercise caution when loading samples in the tray, so as not to force the tray into the instrument or catch ones extremities inside the instrument. To prevent measuring the wrong samples or loading the plate the wrong way, it is critical to orient the plate correctly in the loading tray. Once the plate is set up, it’s time to load the samples. The negative control is a control variable where no measurement/effect is expected to be observed. The positive control indicates whether or not the assay has worked properly. The values obtained from these measurements are called the “background”. The blank is used to determine the extent of your measurement that is not experimentally relevant and is due to the buffers in which your sample is diluted or reagents to which your sample is exposed. This data is then used to create a graph where a line of best fit is generated. ![]() The standard curve uses samples with known concentrations, which yield different absorbance values. Here you see a plate loaded in triplicate. Samples and standards are loaded either in duplicate or triplicate to account for any pipetting errors. Wells can sometimes be loaded using a standard single channel pipette. The reservoirs hold the solutions for the multichannel pipette. ![]() Multichannel pipettes are often used to load multi-well plates. Multiplate readers are used to quantify protein, gene expression and various metabolic processes such as reactive oxygen species and calcium flux. Besides standards and samples being run on the multiwall plate, the blank along with positive and negative controls are also used in the assay to ensure it is working correctly. Experimental values are then extrapolated to the curve or are calculated using the equation from the linear regression. This curve uses samples of known concentration to generate a line of best fit or standard curve. Regardless of the assay type, experiments on the plate reader utilize a standard curve to determine the experimental values. Multiwell plates are integral to the microplate reader and allow for many experiments to be performed at once. Microplate readers can make absorbance, fluorescence and luminescence measurements. The microplate reader is a multimodal instrument that allows for a variety of experiments to be performed and measured simultaneously.
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