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Application Note: ExoTrap™ Exosome Isolation Spin Column Kit for Protein Research

ExoTrap™ Exosome Isolation Spin Column Kit for Protein Research

Reported by Dr. Yoshitaka Sato
Department of Virology, Nagoya University Graduate School of Medicine

Products

  • ExoTrap™ Exosome Isolation Spin Column Kit for Protein Research (Cat. No. CSR-SHI‒EXO‒K010)

Exosomes are 40 ‒100 nm membrane vesicles. They are found in many biological fluids including blood, urine, saliva, amniotic fluid, cell culture supernatants, and malignant ascites fluids. CD9 is a well known surface marker for exosomes. ExoTrap Spin Columns utilize immobilized CD9 antibody to capture exosomes from biological fluids in as little as 30 minutes. The contents of the captured exosomes are then readily processed for downstream analysis by western blotting, mass spectrometry, PCR, or sequencing, etc.

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Experimental Details

 Exosomes are membrane vesicles that are enveloped with a lipid bilayer, and encase various molecular constituents, including proteins and nucleotides. They have a diameter of around 30 - 100nm, and are secreted from various cells. By circulating in body fluids, exosomes can transfer various signals to distant cells. This cell-to-cell communication via exosomes has been thought to be associated with antigen presentation, apoptosis, inflammation, and tumor evolution/malignant alteration, leading to investigation of cancer cell derived exosomes. It is anticipated to apply in a wide range of research area, including development of early detection of cancers using exosomes that are secreted from cancer cells, or drug delivery systems utilizing exosomes as cargo.

We are focusing on investigating the interaction between virus-infected cells and surrounding uninfected cells. After viral protein-expressing cells and non-expressing cells were co-cultured, and virus proteins were detected by fluorescent immunostaining, virus proteins were observed in dot form from the surrounding uninfected cells (the cells that are not expressing viral protein). From this incident, we are interested in exosomes, which intervene cell-to-cell communication.

However, we're facing the problem, "how do you purify exosomes?", since we've never dealt with exosomes by ourselves. In general, the ultracentrifuge method is employed to purify the exosomes, however, it needs to optimize various conditions and is time consuming and laborious when purifying from many samples. For that reason, we compared several exosome isolation kits on the market at some respects, including simple procedure (robustness), required time (quick protocol), and aplenty applications (both the exosome itself and the encapsulated protein or nucleotide can be analyzed). Because of its easy procedure (just apply samples into spin column) and quick protocol (takes only 30min), we chose ExoTrap™ Exosome Isolation Spin Column Kit for Protein Research (Cat No. CSR-SHI‒EXO‒K010) from Cosmo Bio. Ltd.

The advantages of this product is its simpleness, as you just have to apply samples into spin columns and quick spin, and its quick procedure, which takes only 30min. Even though we're "exosome beginners", we could purify not only exosomes but encased virus proteins, which was detected by Western Blot analysis only after column purification. On the other hand, from the conditioned medium of which cells were treated with exosome inhibitor (GW4869), this viral protein wasn't detected suggesting it's still encased by the exosomes (1). This product may not suitable for large sample volumes, however; it is highly recommended when working with small sample volume, and when you prefer a quick and easy exosome purification procedure.


Fig. 1 Results of western blot (WB) analysis with before or after purified exosomes with or without exosome secretion inhibitor treatment.

(A) Exosomes were purified using ExoTrap™ Exosome Isolation Spin Column Kit, and then encased LMP1 and Hsp70 expression were analyzed by WB. Enrichment of LMP1 and Hsp70 in the purified exosomes was observed. (B) Cells were treated with exosome inhibitor (GW4869) and then exosomes were purified from its conditioned medium using ExoTrap™ Exosome Isolation Spin Column Kit. WB was performed to identify LMP1 and Hsp70 expression. The reduction of LMP1 and Hsp70 by the GW4869 treatment was observed, suggesting that those protein are still encased by the exosomes.

Product List

Product Name Cat# Quantity Price

ExoTrap Exosome Isolation Spin Column Kit for Protein Research

CSR-SHI-EXO-K010 10PREP.

¥ 57,000

Reference
  • Sato et al., Oncotarget, 8; 39345‒39355, 2017