How to Analyze Exosome Purification Quality?
- Particle size predominantly within 30–150 nm
- A single-peak or narrow distribution indicating low heterogeneity
- Particle concentration reflecting recovery efficiency
- Positive markers: CD9, CD63, CD81, TSG101, Alix
- Negative markers: Calnexin (endoplasmic reticulum), GM130 (Golgi apparatus), etc.
Exosomes are extracellular vesicles with diameters of approximately 30–150 nm, secreted by a variety of cell types. They play essential roles in intercellular communication and pathological regulation, particularly in research on cancer, neurodegenerative diseases, and immunology. However, extraction approaches such as ultracentrifugation, polymer-based precipitation, and size-exclusion chromatography frequently introduce protein impurities, lipoproteins, or non-exosomal EVs, which can markedly compromise the accuracy and reproducibility of downstream analyses. Therefore, systematic and comprehensive evaluation of exosome purification quality is fundamental for high-confidence omics studies, functional assays, and biomarker development.
Four Core Evaluation Dimensions of Exosome Purification Quality
1. Morphological Assessment: Transmission Electron Microscopy (TEM)
TEM serves as the gold standard for validating characteristic exosome morphology. Negative staining enables clear visualization of the typical “cup-shaped” or “disk-shaped” appearance and allows preliminary identification of membrane damage, cellular debris, or protein aggregates within the sample.
(1) Advantages: Direct visualization and reliable confirmation of exosome structure
(2) Limitations: Low throughput; should be combined with additional characterization methods
2. Particle Size and Particle Concentration: Nanoparticle Tracking Analysis (NTA)
NTA (for example, NanoSight) tracks the Brownian motion of particles in real time to calculate size distribution and particle concentration. It is one of the key technologies for assessing exosome purification quality.
(1) Ideal results:
(2) Recommendation: Combine with TEM to enhance the accuracy of morphological and quantitative assessments
NTA data provide not only particle-size characterization but also an estimate of recovery rate during isolation, enabling quantitative evaluation of exosome purification quality.
3. Exosome Marker Analysis: Western blot / ELISA / Mass Spectrometry
Exosomes are enriched with specific proteins, including:
Detection of these markers by Western blot or ELISA verifies the specificity of exosome enrichment and helps identify contamination originating from cellular organelles, thereby improving the accuracy of purification quality control. Moreover, mass spectrometry provides high-throughput proteomic profiles that support quality assessment and reveal the functional potential and biological origins of exosomes.
4. Purity and Contamination Assessment: Total Protein Amount, RNA Integrity, and Lipoprotein Contamination
(1) Protein-to-particle ratio: Commonly used as an approximate purity indicator. An ideal ratio is below 0.5 μg /10⁹ particles; higher values may indicate contamination by soluble proteins.
(2) Lipoprotein detection: Positive ApoA1 or ApoB suggests possible interference from high-density lipoprotein (HDL) in plasma-derived samples.
(3) RNA quality analysis: Exosomal RNA should consist primarily of small RNAs and lack prominent 28S/18S rRNA peaks. The presence of abundant rRNA indicates contamination from extracellular free RNA or cell lysis.
Together, these analytical approaches establish a quantitative and traceable system for exosome purification quality control, which is crucial for advancing standardized experimental workflows.
High-quality exosome research depends on rigorous and standardized evaluation of exosome purification quality. From morphological confirmation to particle-size profiling, and from marker validation to contamination assessment, each step directly influences data accuracy and the reliability of biological interpretation. This is particularly critical for downstream analyses such as proteomics, metabolomics, and miRNA profiling, where the representativeness and purity of exosomes are decisive factors. MtoZ Biolabs provides comprehensive exosome omics solutions, including isolation, characterization, multi-omics analysis, and bioinformatics support, to help researchers enhance efficiency, ensure data reliability, and accelerate scientific output. If you are planning related projects, you are welcome to contact us for customized technical and data-analysis services.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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