I isolated exosomes form human serum using exosome isolation kit. I lysed the exosome pellet in 1X RIPA buffer with protease inhibitor. I also cultured the fibroblast cells and used the 1XRIPA Buffer to lyse the cell. I ran cell lysate and exosome in 12% polyacrylamide gel and transferred it to PVDF membrane.
I was able to detect CD 63 in exosomes and Calnexin in the cells. Cd 63 was not detected in the cells and Calnexin was not detected in the exosomes sample.
Now I wanted to detect loading control Beta actin and GAPDH in both of the sample, but I am only able to detect beta actin and GAPDH in the cell lysate and not in the exosome sample.
Could anybody answer why I am not able to detect these loading controls in my exosomes.
I tries adding 1X TritonX 100 to the sample, but it did not work?
Thanks
Hello Kamala Lamsal You will not detect Actin in exosomes unless it is being packaged in them. Exosome cargo are variable so using cytoskeleton markers like tubulin and actin is not adequate. As for GAPDH the same principle applies some will have it if it is packaged but it can also attach to outer surface of exosomes which means it may be lost during isolation/lysis procedures (doi.org/10.1038/s41467-021-27056-3). It is best to choose a loading control that is part of the membrane make-up of both cells and exosomes such as Flotillin -1 and Alix or AIP1. Depending on type of cells you are working on CD9 may be an option too. Good luck.
Dear. Dr. Kamala,
Could you please send me in details the protocol of obtaining Exosome lysate for CD63 by western blotting. I used kit for isolation of exosomes from cell culture media . Thanks and all the best.
Hola. Algo así me paso en omvs de bacterias. Puede ser que no contengan las proteínas que están testeando. Probaría con otro método de ruptura de membrana para ver si efectivamente no lo contiene (esto si quiere usar los mismos controles). O seleccionar un componente de membrana que se sabe que está presente en los MV o que es muy abundante en la membrana desde donde provienen. Saludos y que salga bien su experimento.
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