Journal: Molecular & Cellular Proteomics

Article Title: Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform

doi: 10.1074/mcp.m113.032136

Figure Lengend Snippet: FIG. 1. Preparation of highly pure Du145 exosomes for analysis via SOMAscanTM. Du145 exosomal vesicles were separated on a continuous sucrose gradient, and the density of 15 collected fractions was determined. Nanoparticle tracking analysis was performed on each fraction, and the particle concentration was plotted against the fraction density. Bars represent mean S.D. of duplicate measurements (A). The size distribution of particles within each fraction is shown, and the density of each fraction is specified, revealing single-peak, monodisperse populations of small vesicles in fractions of classical exosomal density (between 1.1 and 1.2 g/ml) (B). A proportion of each fraction was coated onto latex microbeads, stained with antibodies as specified, and analyzed via flow cytometry. Bars represent median fluorescence values from 5000 events, and the positions of fractions 8–10 are annotated (C). This characterization aided in selecting relevant fractions—specifically, F8, F9, and F10—that were pooled for subsequent array analyses.

Article Snippet: Immuno-blotting—Lysates of sucrose cushion-purified Du145 exosomes or whole cells, made using RIPA buffer (Santa Cruz), were boiled in SDS sample buffer containing 20 mM DTT as previously described (29) and prepared as matched protein doses (12.5 g to 50 g per lane).

Techniques: Concentration Assay, Staining, Flow Cytometry, Fluorescence

Journal: Molecular & Cellular Proteomics

Article Title: Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform

doi: 10.1074/mcp.m113.032136

Figure Lengend Snippet: FIG. 4. Confirmation of the expres- sion of selected SOMAscanTM-identi- fied proteins. Du145 exosomes purified via the sucrose cushion method were immobilized at specified doses on ELISA microplates and probed using individual SOMAmers (A) or an indirect staining method with antibodies (B). Signals were detected using Europium-streptavidin and time-resolved fluorimetry (TRF) in each case (bars represent mean S.E. of duplicate measurements). SOMAmers (KDGL or Spuriomer) act as irrelevant controls for nonspecific binding (A). To confirm that the identified proteins float at a classical exosomal density, a con- tinuous sucrose gradient fractionation was performed, and the density of col- lected fractions was determined prior to immobilization on microplates as de- scribed above. Proteins were detected with antibodies using the same indirect staining method as described above. The fractions with densities between 1.1 and 1.2 g/ml are annotated (C). Whole cell lysates and exosomes normalized for protein were subjected to SDS-PAGE and Western blotting and probed with antibodies as indicated. This revealed relative exosomal enrichment for all can- didates, whereas calnexin exhibited the reverse pattern (D). Sucrose cushion-pu- rified exosomes were labeled in solution with primary antibody (as specified) and secondary biotinylated antibody before being immobilized on microplates pre- coated with anti-CD9 or isotype control antibody. After washing, signals were detected using Europium-streptavidin and TRF (bars represent mean S.E. of quadruplicate measurements) (E).

Article Snippet: Immuno-blotting—Lysates of sucrose cushion-purified Du145 exosomes or whole cells, made using RIPA buffer (Santa Cruz), were boiled in SDS sample buffer containing 20 mM DTT as previously described (29) and prepared as matched protein doses (12.5 g to 50 g per lane).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining, Binding Assay, Fractionation, SDS Page, Western Blot, Labeling, Control