Western blot analysis of exosomes isolated from MM and colostrum. (a) Western blot analysis of fractions of increasing density obtained from the OptiPrep density gradient centrifugation which were probed for exosomal markers Alix and TSG101. TSG101 was identified in all the samples from fractions 5–9 corresponding to the buoyant density of 1.08–1.22 g/mL. The presence of Alix was limited to colostrum samples only. (b) Western blot analysis revalidating the presence of Alix in higher abundance only within colostrum exosomes as compared to mature milk (MM) and commercial milk (CM). TSG101 was found in all the samples including commercial milk exosomes. Exosomes isolated from LIM1215 colorectal cancer cells (LIM1215) was used as a positive control. GM130 was detected in LIM1215 colorectal cancer cell lysates (WCL) and was absent in the milk derived exosomal samples confirming the depletion of contaminating vesicles arising from apoptosis.