Can Flow Cytometry Achieve Absolute Protein Quantification? What Methods Accurately Measure Two Membrane Proteins per Cell?
Flow cytometry is a powerful technique for analyzing and sorting cells based on specific markers on their surface or within the cytoplasm. While it is highly effective for qualitative assessment and relative quantification—allowing comparisons of protein expression levels across different samples—it is generally not used for absolute quantification. To achieve accurate quantification of membrane proteins, particularly to determine the absolute number of molecules on the surface of a single cell, the following approaches can be employed:
Mass Spectrometry-Based Quantification
Mass spectrometry offers robust methods for absolute protein quantification, such as multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM). These techniques, when combined with internal standards of known concentrations, enable the precise measurement of protein copy numbers at the molecular level.
Single-Molecule Techniques
Advanced single-molecule approaches—such as single-molecule fluorescence microscopy or single-molecule localization microscopy—allow direct visualization and counting of individual protein molecules on the cell surface. While highly accurate, these methods typically require sophisticated imaging platforms and rigorous data analysis, making them resource-intensive and time-consuming.
Together, these methodologies complement flow cytometry and provide feasible solutions for achieving absolute quantification of membrane proteins at the single-cell level.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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