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.