Qiagen, 76204, Buffer XBP (250 ml)

250 ml Binding Buffer

Features

- Isolate intact exosomes and other extracellular vesicles
- Suitable for functional testing and physical and biochemical analysis
- Purify exosomes and other extracellular vesicles in 25 minutes
- Process multiple samples in parallel using a simple spin-column procedure
- Use with up to 4 ml of plasma or serum or 32 ml of cell culture supernatant
- Suitable for MISEV-compliant protein characterization

Performance

The exoEasy protocol for purification of exosomes and other extracellular vesicles is not only faster than ultracentrifugation, but also yields a cleaner preparation. Scanning electron microscopy shows that while both techniques deliver intact vesicles of the expected size, the preparation from ultracentrifugation also contains many smaller structures that do not match the expected size or shape for EVs (see figure “ Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation ”).

Fully intact, spherical vesicles of different sizes between approximately 50-300 nm diameter can also be observed using cryo-electron microscopy. (see figure " Visualization of EVs isolated using the exoEasy Kit by cryo-electron microscopy "). Isolated vesicles are fully functional and can be used for several downstream applications including cell culture work (see figure “ Extracellular vesicles isolated using the exoEasy Kit are taken up efficiently by target cells (customer data) ”). Quality and quantity of eluted vesicles can be determined by a range of different methods. These include electron microscopy, physical characterization (e.g., Nanoparticle Tracking Analysis (NTA), or Tunable Resistive Pulse Sensing (TRPS)) and analysis of molecular constituents of vesicles, such as proteins, nucleic acids or lipids. Since particle counting techniques like NTA or TRPS do not distinguish vesicles from other particulate matter, particle counts performed in unprocessed biological fluids or crude preparations that still contain other particulate matter (e.g., protein complexes) may vastly overestimate the number of vesicles present.

To standardize protocols and reporting in the EV field, the International Society of Exosomes has developed the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. These guidelines provide examples of EV-enriched protein markers that can be used to characterize EVs. However, analyzing protein expression in intact EVs can be challenging due to the limited amount of material and low expression levels of many EV proteins.

In partnership with ProteinSimple (a Bio-Techne brand) QIAGEN has developed a standardized workflow for EV protein characterization. This new workflow combines a simple method for isolating EVs with automated protein separation and immunodetection of MISEV-recommended proteins. Following isolation with the exoEasy Maxi Kit, protein expression of intact EVs can be characterized on Simple Western by ProteinSimple. Simple Western is a western blotting platform for automated protein separation and immunodetection. Signals from potentially contaminating proteins such as APOA1 are efficiently depleted during isolation. (See figure “ EV protein analysis across plasma fractions using Simple Western automated western blot ”).

Applications

- Physical characterization (e.g., NTA, TRPS)
- Electron microscopy
- Isolation and analysis of DNA, RNA*, proteins, lipids and other constituents
- Flow cytometry
- Antibody- or fluorescent dye-based labeling
- Uptake by recipient cells