Journal: Proteomes

Article Title: Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma

doi: 10.3390/proteomes7020017

Figure Lengend Snippet: EV analysis by western blotting. Proteins from crude body fluids (blood plasma, PL, ( A ); cerebrospinal fluid, CSF, ( B ); seminal plasma, SP, ( C ) of individual animals as well as extracellular vesicles (EV) isolated from such biofluids were separated by SDS-PAGE and blotted to detect the EV markers Alix (110 kDa) and TSG101 (44 kDa). Porcine IgG heavy chains highly abundant in blood plasma were largely removed during EV isolation. The membrane was silver stained to detect protein loads in all lanes ( D ). Positive controls included human H9-derived neural stem cell line (H9) and porcine tissues (B = brain, T = testes). M = molecular weight standard. Representative images from at least three experiments are shown.

Article Snippet: Anti-Alix 1:1500 (Abcam, ab88388, Cambridge, UK), anti-TSG101 1:500 (Origene, TA333922, Rockville, MD, USA), anti-β-tubulin 1:2000 (Merck, T4026, Kenilworth, NJ, USA), anti-UQCRC1 1:250 (Merck, HPA002815, Kenilworth, NJ, USA), anti-CD63 1:1000 (Santa Cruz Biotechnology, sc5275, Dallas, TX, USA), antibodies were diluted in 5% milk in TTBS, anti-lamin A/C 1:2000 (Merck, SAB4200236, Kenilworth, USA) in 1% milk, and anti-CD9 1:500 (Aviva, ARP61171, London, UK), anti-CD9 1:20 (Sysmex, CP638591, Kobe, Japan), and anti-CD63 1:200 (Exbio, clone MEM-259, Prague, Czech Republic) in 5% BSA, followed by peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse IgG, Jackson ImmunoResearch 711-035-152 and 715-035-151, resp., as appropriate, West Grove, PA, USA) used for specific protein detection.

Techniques: Western Blot, Isolation, SDS Page, Staining, Derivative Assay, Molecular Weight