Irdye 800cw conjugated goat anti rabbit secondary antibody

Pooled human serum (Valley Biomedical, Winchester, VA, catalog #HS1004P) and six commercially available individual donor samples (BioreclamationIVT, NY, Item #HMSRM) were used for exosome isolation. We used three exosome isolation kits for this study: miRCURY^TM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuick^TM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA). To isolate exRNA, miRCURY RNA Isolation Kit–Cell & Plant (Exiqon, Woburn, MA) was used. RIPA lysis and extraction buffer (GBiosciences, St. Louis, MO), 2xLaemelli buffer (Bio-Rad, Hercules, California), and 100x halt protease inhibitor EDTA-free cocktail (Thermo Scientific, Grand Island, NY) were used to prepare protein samples for western blot analysis. Rabbit monoclonal anti-CD63 antibody was from Abcam (Cambridge, MA), and rabbit polyclonal anti-CD9 antibody was purchased from Santa Cruz (Dallas, Texas). IRDye 800CW-conjugated goat anti-rabbit secondary antibody was from LI-COR (Lincoln, Nebraska).

Pellets containing a mix of embryos, larvae and adults were prepared as described above. Lysates were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in lysis buffer (50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl₂, 0.1% Triton X-100, 5% glycerol (w/vol), 1 mM PMSF, 7 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001). Lysates were cleared by centrifugation at 16100 x g for 20 minutes at 4°C. Protein concentrations were approximated by Bradford Assay (Bio-Rad). Samples were prepared by mixing 25 micrograms of protein extract with the required amount of 4x NuPAGE™ LDS Sample Buffer (Invitrogen, NP0007) and 10x NuPAGE Sample Reducing Agent (Invitrogen, NP0004), followed by heating at 70°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane by wet-transfer. The following primary antibodies were used: 1:1000 monoclonal mouse anti-eEF1A (Merck, 05–235), 1:10000 monoclonal mouse anti-puromycin (Merck, MABE343), 1:5000 polyclonal rabbit anti-actin (Abcam, ab8227). Detection was carried out with IRDye 680RD-conjugated goat anti-mouse secondary antibody (LI-COR Biosciences, 926–68070) or IRDye 800CW-conjugated goat anti-rabbit secondary antibody (LI-COR Biosciences, 926–32211) and infrared imaging (LI-COR Biosciences, Odyssey CLx).

The expression of EGFP was evaluated by Western blot. The 10 μL of supernatant was loaded into each well of 10% SDS-PAGE gel. After finishing the electrophoresis, the proteins in the gel were transferred to Hybond-C nitrocellulose membrane (Amersham Bioscience). The transfer was done at 100 V for 2 h. Anti-EGFP antibody (Proteintech, China, Cat no. 50430-2-AP) and IRDye 800CW-conjugated goat anti-rabbit secondary antibodies (LI–COR Biosciences, Lincoln, NE, USA; cat. no. C60607-15) were employed as the primary and secondary antibody, respectively. The hybridization signals were detected and measured using LICOR Odyssey system (LI–COR, Nebraska, USA).

The expression of reporters was analyzed by Western blot. Briefly, 10 μl of supernatant was separated on 12% SDS-PAGE. After transferring the proteins in gel to Hybond-C nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK), the NC membranes were blocked with 5% fat-free milk and incubated overnight at 4 °C with anti-His tag antibody (Sangon Biotech, China, Cat no. D191001). IRDye 800CW-conjugated goat-anti-Rabbit secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA; cat. no. C60607-15) was employed to detect the hybridization signal at 1:1000 dilutions. The signals were measured using LICOR Odyssey system (LI-COR, Nebraska, USA).

Harvested cells were washed once with 1-ml 1 M sorbitol, and the cells were resuspended by adding 100 μl breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM PMSF, 1 mM EDTA and 5% glycerol). An equal volume of acid-washed glass beads (Sigma; Cat no. G8772) was added to the resuspension. The mixture was vortexed for 30 s and then immediately placed on ice for 30 s. This operation was repeated eight times. The cell lysate was transferred to a new tube after centrifuging at 16,000 rpm for 10 min at 4 °C. Protein concentration was quantified by using Pierce™ BCA Protein Assay Kit (Thermo; Cat no. 23227) and then boiled and denatured at 95 °C for 5 min. For western blot analysis, the 30 μg samples were separated on 10% SDS-PAGE, and protein bands were transferred to Hybond-C nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK) through electroblotting. The membranes were blocked with 5% fat-free milk and probed overnight at 4 °C with primary antibody, against EGFP (Proteintech, China, Cat no. 50430-2-AP), and IRDye 800CW-conjugated goat anti-rabbit secondary antibodies (LI–COR Biosciences, Lincoln, NE, USA; cat. no. C60607–15) were used as the secondary antibody at 1:1000 dilutions. The signals were detected and measured using LICOR Odyssey system (LI–COR, Nebraska, USA).