Anti β casein ab112595

Raw bovine milk was collected from a local farm (Madrid, Spain). Milk-derived EXOs were isolated and purified as previously described [23 (link)], using sequential centrifugation, ultracentrifugation, and size exclusion chromatography (SEC) steps. The protein concentration of each eluted fraction after SEC was determined by the BCA method (Thermo Scientific, Waltham, MA, USA), using BSA as the standard and following the manufacturer’s instructions. The identification of EXOs was assessed by Western blot (WB) analysis using anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, Bioss, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl), and anti-β-casein (ab112595, Abcam, Cambridge, UK) as primary antibodies. Anti-rabbit or anti-mouse conjugated secondary antibodies were used with either Alexa Fluor^TM 680 or IRDye^® 800. Blots were visualized using an Odyssey^® infrared imaging system (LI-COR, Lincoln, NE, USA) and analyzed for image processing with the Image Studio Lite 5.2.5 software (LI-COR) [23 (link)].

To determine the presence of exosomes, 20 µL of each fraction or 50 µg/protein of each sample were used for protein detection. Protein samples lysed in reducing conditions were separated on 10% sodium dodecyl polyacrylamide/bisacrylamide gels, transferred onto nitrocellulose membranes (0.22 µm Millipore) and blocked with 2.5% skimmed milk (Bio-Rad, Hercules, CA, USA), for 30 min, at room temperature. Membranes were then incubated with appropriate primary antibodies: anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, BIOSS, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl) or anti-β-casein (ab112595, abcam, Cambridge, UK) at 4 °C (overnight). After incubation, membranes were exposed to secondary anti-rabbit or anti-mouse conjugated antibodies, with either Alexa FluorTM 680 or IRDye^® 800, for 45 min at room temperature. Blots were washed three times with TBST buffer after each incubation step and visualized using an Odyssey^® infrared imaging system (LI-COR, Lincoln, NE, USA). Image Studio Lite 5.2.5 software was employed for image processing.