I work on exosomes isolated (via ultracentrifugation) from MDA-231 breast cancer cells.
When I run westerns to confirm isolation I generally use Alix and Flotillin - these are antibodies I borrowed from another researcher in my department. I get some mixed results - the Alix signal is general weak if present at all. I also realized the Flotillin ab I have is for Flotillin-2 and not Flotillin-1, the more classical exosomal marker (at least in papers I have read) - are both of these proteins supposed to be present in/on isolated exosomes?
In terms of recommended proteins to blot for I have seen -
Flotillin-1
Alix
TSG101
CD9
CD63
CD81
HSC70
Are some of these markers more enriched in breast cancer lines/exosomes than others? I cannot purchase all the antibodies at once and am hoping to prioritize by what is most likely to be successful.
I'm also curious about what kinds of protein yields other people are getting from ultra-centrifugation isolation. I generally get only around 3ug of protein total - this is modest considering the starting material is 100ml of conditioned media. The low protein yield may relate to the inconsistencies I am experiencing with Alix and Flot-2.
In Summary - What markers have people had success with when performing western blots on MDA-231 (or other breast/epithelial cancers) derived exosomes? and how much exosomal protein was loaded to detect consistent banding?
CD63 works very well, though it would also be interesting to add a specific surface marker of your cell type so that you can confirm their origin. Also, if you want to buy any antibody, and have where to choose, cell signaling is a brand that usually gives good results...
Hello,
talking about breast cancer cell lines, among the most used exosomes markers there are HSP70 and HSP-90. See for example this paper where they used also MDA-231:
`Exosomes Released from Breast Cancer Carcinomas Stimulate Cell Movement`.
In terms of the molecular weight and the absence of non-specific bands, CD63 seems to be the most reliable marker for WB analysis of exosomes.
CD63 works very well, though it would also be interesting to add a specific surface marker of your cell type so that you can confirm their origin. Also, if you want to buy any antibody, and have where to choose, cell signaling is a brand that usually gives good results...
is there any specific protein marker that distinguish microvesicles from exosomes.
Most popular exosomes markers are:
1. CD63
2. CD9
3. CD81
4. MDA231
5. HSP70
6. HSP90
Hello Will Kietzman
Did you fix your problems? I'm asking because I have similar troubles. It seems that MDA231 cells secrete fewer exosomes than other breast cancer cell lines. I need to start with minimum 100-150 ml of conditioned medium. But, I still have problems to see exosomal markers in western blot.
Can you help me with some tip?
Thanks a lot!!
Hi Eduardo Duran
Did you use Alix for MDA-231? If yes, Alix is not a suitable marker of exosomes.https://www.nature.com/articles/s42003-019-0570-8
You may wish to try Flotillin, I have seen a high signal of flotillin, CD9 expression in 231.
One way to increase the yield of exosome protein concentration is to seed a large number of cells in relatively small conditioned media. In this way, exosomes for download stream analysis will be efficient and visible for detection.
Also, you can try to lyse the exosomes to break down the vesicle membrane since most proteins are inside the cargoes.
Hi Abayomi Opadele , I tried Alix a couple of times, but it didn't work. Currently I'm uisng CD63, TSG101 and Flotilin-1
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