Purification and analysis of endogenous human exosomes. (A) (1) 3xFlag-tagged exosome component is affinity captured using anti-Flag antibodies coupled to magnetic beads. Pink and blue circles represent proteins not related to the exosome. (2) Native elution is performed with 3xFlag peptide. (3) Eluted complexes are further fractionated on a glycerol gradient and analyzed as indicated. (B) Silver stained SDS polyacrylamide gel displaying the fractionation of ExoI on a 10%–40% glycerol gradient. Exosome constituents are labeled; bands marked “core” consist of the low molecular mass components EXOSC1-8, MPHOSPH6, and C1D. Black arrow indicates the peak fraction. (C) Peak fractions from ExoI and ExoII glycerol gradients analyzed by SDS-PAGE. The ExoI peak fraction was stained with silver, ExoII with Sypro Ruby. Protein bands are labeled as in B. (D) MS-based estimate of the relative amounts of EXOSC10 and DIS3 obtained in velocity sedimented fractions of ExoI and ExoII preparations, as in C. Error bars indicate the data range. (E) Negative-stain TEM analysis of ExoI and ExoII particles. Shown are 16 representative 2D class averages for each preparation. Scale bars (black): 10 nm. (F) ExoI and ExoII 2D class averages have been enlarged to illustrate the “hole,” observed in ExoI class averages and the “lobe,” observed in ExoII class averages (white arrows).
