In general, given the complexities and unique characteristics of different biological samples, protocols for purification and characterisation must be optimised. In the case of extracellular vesicles (EVs), the presence of non-EV particles such as proteins and larger particles (e.g., cells and cell fragments) can prove problematic for TRPS measurements. This mostly occurs via blocking of the nanopore by contaminating particles which are too large for the nanopore, and the ‘sticky’ environment created by high protein contamination. If you encounter frequent blockages when running your EV samples, it is therefore important to ensure your sample processing is optimised.
To remove large particles, sequential centrifugation or dead-end filtration can be used.
For biofluids with lower levels of EVs and proteins, the sample can be concentrated (e.g., using tangential flow filtration) to enable larger volumes to be processed (not applicable to serum and plasma that have very high levels of protein and cannot be readily concentrated). The exact protocol may require modification depending on the sample, its viscosity and other preparation steps.
For the EV isolation itself, we recommend our size exclusion chromatography qEV Gen 2 columns, which isolate EVs whilst removing ~99% of protein contamination. Concentration can additionally be done following EV isolation if required.
These steps should produce an isolate which is sufficiently clean for TRPS. Our support team have found that switching to qEV isolation from alternative methods have aided customers having trouble with TRPS measurements. If this does not improve your measurements, please get in touch with our support team who will be able to aid you further.
Izon provides example protocols for preparation of extracellular vesicles from common sample types:
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