These small (30-150nm) extracellular vesicles aren’t cellular “trash bags” after all. Exosomes are now believed to be present in all body fluids, where they are thought to play a role in a large number of biological functions.
What’s the mechanism of exosome formation? What is their composition? How can they best be isolated and studied? The field of exosomes research is truly captivating, with an urgent need for a range of specific tools and technologies.
A range of specific tools and technologies are needed for the isolation and analysis of exosomes and their cargo. The products presented here have been developed to simplify the overall study of exosomes, their composition, and function.
Ultracentrifugation can be tedious and time consuming. Now you can easily enrich for intact exosomes from a variety of starting samples using the flexible and scalable Total Exosome Isolation reagents. These reagents and accompanying protocols are ideal for a range of experimental set-ups including low volume input and handling of multiple samples.
Total exosomes enriched from cell culture (by using the Total Exosome Isolation reagents or ultracentrifugation) can be further purified into specific subpopulations by immunomagnetic capture. Use the Dynabeads®-based CD63-specific reagent, or combine the Streptavidin reagent with your choice of a biotinylated antibody to purify any population based on a surface antigen.
|- Molecular assays (e.g. PCR, Western)
- Biological assays (e.g. tracing exosomes in vivo)
- Intact exosome analysis (e.g. flow and EM)
|- Molecular assays
- Intact exosome analysis
Visual identification of exosomal vesicles can be challenging, and free exosomes alone are too small to be detected in flow. There are a few methods, though, which can be used for analysis.
Exosomes recovered with the Total Exosome Isolation reagent can be analyzed using the NanoSight® instrument for approximate size range and concentration.
Exosomal RNA or membrane components can be labeled, allowing for visualization of exosomes under the microscope. Specialized spin columns are available to remove unincorporated dyes. These columns also allow for buffer exchange and easier desalting or removal of low molecular weight (< MW 3000) contaminants compared to current methods.
Electron Microscopy (EM)
One of the major advantages of employing Dynabeads® magnetic separation technology to pull out pure and specific exosomes is that you can move directly to EM analysis. You can analyze subsets from total exosomes enriched from cell culture using the CD63-specific reagent, or use the Streptavidin reagent in combination with your choice of biotinylated antibody.
Immunomagnetic capture of exosomes on the surface of the Dynabeads® allow for a clear and defined FFC/SSC detection typically in less than one hour. You can analyze subsets from total exosomes enriched from cell culture using the CD63-specific reagent, or use the Streptavidin reagent in combination with your choice of biotinylated antibody.
Exosomes have been shown to transport a range of molecules from one cell to another. Their cargo includes proteins, lipids, mRNA (fragments and full length), rRNA, miRNA, and various ncRNA.
RNA & Protein Isolation
Simultaneous isolation of both proteins and total RNA from the same sample of pre-enriched exosomes can be achieved using the Total Exosome RNA and Protein Isolation Kit. The kit is compatible with all protocols for exosome isolation.
Exosomal proteins can be analyzed using Western blot analysis. If you are looking to compare multiple protein samples on the same gel and want to keep exosomal protein complexes intact, you should check out the fast and gentle Dynabeads®-based immunoprecipitation method using Protein A or Protein G in combination with your primary antibody of choice.
"Exosomes-The Next Small Thing"
Don’t miss any of the episodes:
The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo Schageman J, et. al. BioMed Research International 2013, doi:10.1155/2013/253957
Methods for the extraction and RNA profiling of exosomes Zeringer E, et al. World J Methodol 2013 March 26; 3(1): 11-18
Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Biochim Biophys Acta. 2012 Jul;1820(7):940–-8