For western blot analysis, ultracentrifuged exosomal pellets were lysed with RIPA buffer (Cell Signaling Technology) containing a Protease Inhibitor Cocktail (Calbiochem). The total protein was determined using a BCA kit, and an equivalent amount (25 μg) of exosomal protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed overnight at 4°C with antibodies specific for Alix, Tsg101, HA, GM130 as indicated25 (link). IRDye IgG was used as secondary antibody (1:10,000) for 30 min. Bands were visualized on a Li-COR Odyssey Infrared Imager (Li-COR).
Protocol detail
Exosome Protein Characterization by Western Blot
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Antibodies
Antibody
Buffer
Membranes
Nitrocellulose
Pellets
Protease inhibitor
Protein
Ripa
Sds page
Tsg101
Western blot analysis
Corresponding Organization : Nanjing University
Other organizations : Model Animal Research Center, Nanjing University of Chinese Medicine
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Variable analysis
independent variables
- Exosomal pellets were lysed with RIPA buffer containing a Protease Inhibitor Cocktail
- Protein expression levels of Alix, Tsg101, HA, GM130 as measured by western blot analysis
- Total protein amount (25 μg) used for western blot analysis
- Secondary antibody (IRDye IgG) used for detection
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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