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Western blot analysis of relative protein expression relies on appropriate reference proteins for data normalization. Small extracellular vesicles (sEVs), or exosomes, are increasingly recognized as potential indicators of the physiological state of cells due to their protein composition. Therefore, accurate relative sEVs protein quantification is crucial for disease detection and prognosis applications. Currently, no documented ubiquitous reference proteins are identified for precise normalization of a protein of interest in sEVs. Here we showed the use of total protein staining method for sEVs protein normalization in western blots of samples where conventional housekeeping proteins like β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are not always detected in the sEVs western blots. The No-Stain™ Protein Labeling (NSPL) method showed high sensitivity in sEVs-protein labeling and facilitated quantitative evaluation of changes in the expression pattern of the protein of interest. Further, to show the robustness of NSPL for expression analysis, the results were compared with quantitative mass spectroscopy analysis results. Here, we outline a comprehensive method for protein normalization in sEVs that will increase the value of protein expression study of therapeutically significant sEVs.

No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins