Protein Level Quantification Across Fluorescence-based Platforms
In: Bio Protoc, 2023
academicJournal
Zugriff:
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.
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Protein Level Quantification Across Fluorescence-based Platforms
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Autor/in / Beteiligte Person: | Romero, Hector ; Schmidt, Annika ; Cardoso, Cristina M. |
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Zeitschrift: | Bio Protoc, 2023 |
Veröffentlichung: | Bio-Protocol, 2023 |
Medientyp: | academicJournal |
DOI: | 10.21769/BioProtoc.4834 |
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