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Isolation of Extracellular Vesicles from human plasma samples: The importance of controls
Isolation of Extracellular Vesicles from human plasma samples: The importance of controls
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Length:
20 minutes
Released:
Nov 2, 2022
Format:
Podcast episode
Description
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.31.514442v1?rss=1
Authors: Lazaris, A., Metrakos, P., Petrillo, S.
Abstract:
There are a number of methods for the isolation of extracellular vesicles (EV) which include the traditional ultracentrifugation to column-based kits available from different companies. Isolation of EVs from complex fluids, such as blood, has several challenges as the detection of low abundance molecules can easily be masked by more abundant proteins, when performing mass spectrometry. For this reason, several commercially available kits contain Thromboplastin D (TP-D) to promote clotting, thus removing clotting factors and abundant proteins resulting in increased detection of proteins. Our study demonstrates that plasma pretreated with Rabbit brain derived TP-D (the most common additive), generated a dynamic range of proteins compared to plasma alone, however, most of these proteins were contaminants introduced from the TP-D (99.1% purity). As an alternative, we tested recombinant TP and demonstrated that although it did not introduce any significant contaminants, we did not see any difference in the detection of proteins. Thus TP-D is not required, and any protein additives must be carefully screened.
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Podcast created by Paper Player, LLC
http://biorxiv.org/cgi/content/short/2022.10.31.514442v1?rss=1
Authors: Lazaris, A., Metrakos, P., Petrillo, S.
Abstract:
There are a number of methods for the isolation of extracellular vesicles (EV) which include the traditional ultracentrifugation to column-based kits available from different companies. Isolation of EVs from complex fluids, such as blood, has several challenges as the detection of low abundance molecules can easily be masked by more abundant proteins, when performing mass spectrometry. For this reason, several commercially available kits contain Thromboplastin D (TP-D) to promote clotting, thus removing clotting factors and abundant proteins resulting in increased detection of proteins. Our study demonstrates that plasma pretreated with Rabbit brain derived TP-D (the most common additive), generated a dynamic range of proteins compared to plasma alone, however, most of these proteins were contaminants introduced from the TP-D (99.1% purity). As an alternative, we tested recombinant TP and demonstrated that although it did not introduce any significant contaminants, we did not see any difference in the detection of proteins. Thus TP-D is not required, and any protein additives must be carefully screened.
Copy rights belong to original authors. Visit the link for more info
Podcast created by Paper Player, LLC
Released:
Nov 2, 2022
Format:
Podcast episode
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