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Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus

Western blot detection (a) and relative quantification (b) of characteristic EV markers in PROSPR-separated EV samples after treatment with a range of varying doses of SDS detergent.Significantly higher amounts of CD63, CD9 and Alix proteins were detected at SDS concentrations ≥5% in the lysis buffer (*p < 0.05). The SDS dose-dependent western blots of plasma EVs were performed in triplicate. The stability of EVs was also assessed by western blot (c) and no clear differences between the time intervals studied were revealed. These data suggest that the isolated EVs remain stable for at least four hours in the PROSPR buffer and require higher concentrations of solubilizing agent to be properly lysed.
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f1: Western blot detection (a) and relative quantification (b) of characteristic EV markers in PROSPR-separated EV samples after treatment with a range of varying doses of SDS detergent.Significantly higher amounts of CD63, CD9 and Alix proteins were detected at SDS concentrations ≥5% in the lysis buffer (*p < 0.05). The SDS dose-dependent western blots of plasma EVs were performed in triplicate. The stability of EVs was also assessed by western blot (c) and no clear differences between the time intervals studied were revealed. These data suggest that the isolated EVs remain stable for at least four hours in the PROSPR buffer and require higher concentrations of solubilizing agent to be properly lysed.

Mentions: Since detailed biochemical analysis of EV contents requires membrane disaggregation and release of vesicular cargoes, we next assessed the efficiency of EV disruption by Western Blot with a range of different SDS concentrations (1–10% SDS in PBS). EV lysis was determined by detection of common EV markers (Fig. 1a,b). We observed that maximal detection of the EV-associated proteins CD9, CD63, and Alix was achieved using SDS concentrations ≥5%. Our data further indicated that higher SDS concentrations were associated with improved release of total EV proteins, consistent with earlier reports that ultracentrifugation-separated EVs include distinct subsets with detergent-resistant properties13. We next assessed the stability of the plasma EVs over time when plasma was incubated in PROSPR precipitation buffer at −20 °C up to four hours. Western blot determination of CD9 levels clearly demonstrated that EVs prevailed during the time interval assessed in acetone precipitation buffer, with no significant differences in antigen detection (Fig. 1c). Finally, we assessed the relative yielding profile of PROSPR by western blot (Fig. 2a,b). The results showed that PROSPR isolated between the 50%–70% of the whole amount of EV characteristic antigens from blood plasma. These data collectively suggest that blood plasma EVs can be successfully isolated by acetone-based PROSPR separation and remain stable in PROSPR buffer during at least 4 hours, furthermore these EVs can only be efficiently lysed using high concentrations of denaturing agents.


Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

Gallart-Palau X, Serra A, Wong AS, Sandin S, Lai MK, Chen CP, Kon OL, Sze SK - Sci Rep (2015)

Western blot detection (a) and relative quantification (b) of characteristic EV markers in PROSPR-separated EV samples after treatment with a range of varying doses of SDS detergent.Significantly higher amounts of CD63, CD9 and Alix proteins were detected at SDS concentrations ≥5% in the lysis buffer (*p < 0.05). The SDS dose-dependent western blots of plasma EVs were performed in triplicate. The stability of EVs was also assessed by western blot (c) and no clear differences between the time intervals studied were revealed. These data suggest that the isolated EVs remain stable for at least four hours in the PROSPR buffer and require higher concentrations of solubilizing agent to be properly lysed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588595&req=5

f1: Western blot detection (a) and relative quantification (b) of characteristic EV markers in PROSPR-separated EV samples after treatment with a range of varying doses of SDS detergent.Significantly higher amounts of CD63, CD9 and Alix proteins were detected at SDS concentrations ≥5% in the lysis buffer (*p < 0.05). The SDS dose-dependent western blots of plasma EVs were performed in triplicate. The stability of EVs was also assessed by western blot (c) and no clear differences between the time intervals studied were revealed. These data suggest that the isolated EVs remain stable for at least four hours in the PROSPR buffer and require higher concentrations of solubilizing agent to be properly lysed.
Mentions: Since detailed biochemical analysis of EV contents requires membrane disaggregation and release of vesicular cargoes, we next assessed the efficiency of EV disruption by Western Blot with a range of different SDS concentrations (1–10% SDS in PBS). EV lysis was determined by detection of common EV markers (Fig. 1a,b). We observed that maximal detection of the EV-associated proteins CD9, CD63, and Alix was achieved using SDS concentrations ≥5%. Our data further indicated that higher SDS concentrations were associated with improved release of total EV proteins, consistent with earlier reports that ultracentrifugation-separated EVs include distinct subsets with detergent-resistant properties13. We next assessed the stability of the plasma EVs over time when plasma was incubated in PROSPR precipitation buffer at −20 °C up to four hours. Western blot determination of CD9 levels clearly demonstrated that EVs prevailed during the time interval assessed in acetone precipitation buffer, with no significant differences in antigen detection (Fig. 1c). Finally, we assessed the relative yielding profile of PROSPR by western blot (Fig. 2a,b). The results showed that PROSPR isolated between the 50%–70% of the whole amount of EV characteristic antigens from blood plasma. These data collectively suggest that blood plasma EVs can be successfully isolated by acetone-based PROSPR separation and remain stable in PROSPR buffer during at least 4 hours, furthermore these EVs can only be efficiently lysed using high concentrations of denaturing agents.

Bottom Line: Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR).PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension.We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, 637551.

ABSTRACT
Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

No MeSH data available.


Related in: MedlinePlus