Protein Level Quantification Across Fluorescence-based Platforms

Abstract

Biological processes are dependent on protein concentration and there is an inherent variability among cells even in environment-controlled conditions. Determining the amount of protein of interest in a cell is relevant to quantitatively relate it with the cells (patho)physiology. Previous studies used either western blot to determine the average amount of protein per cell in a population or fluorescence intensity to provide a relative amount of protein. This method combines both techniques. First, the protein of interest is purified, and its concentration determined. Next, cells containing the protein of interest with a fluorescent tag are sorted into different levels of intensity using fluorescence-activated cell sorting, and the amount of protein for each intensity category is calculated using the purified protein as calibration. Lastly, a calibration curve allows the direct relation of the amount of protein to the intensity levels determined with any instrument able to measure intensity levels. Once a fluorescence-based instrument is calibrated, it is possible to determine protein concentrations based on intensity.Key features• This method allows the evaluation and comparison of protein concentration in cells based on fluorescence intensity.• Requires protein purification and fluorescence-activated cell sorting.• Once calibrated for one protein, it allows determination of the levels of this protein using any fluorescence-based instrument.• Allows to determine subcellular local protein concentration based on combining volumetric and intensity measurements.Graphical overview Protein level quantification across fluorescence-based platforms