I am isolating exosome from cancer cells and doing SEM/TEM pretty well. But I am finding it very hard to extract protein from exosomes. Can anyone suggest a good protocol where I can extract total exosomal protein without using any kit.?
Thank you in advance.
Hello Faruk,
Can you tell me, from how many cells you are isolating exosomes to be used for protein isolation? I have a pretty good experience with secretome and trust me that's really tough task. May be I can help you out.
Cheers!
My cell number is around 1-2*10^6..Ya its very tough. Can you help me please..?? Ajay Kumar
Dear Faruk,
Cell number is very low..As there is very low amount of protein which is secreted by a cell...you need to start with very high number of cells. Can you grow some 6×10^7 cells? Is it possible for you to culture so many cells and isolate exosomes?
Cheers !
Hello Faruk,
sorry for responding late, do culture your cells to a large scale as protein is in very low amount in exosomes. Culture around 10-T75 flasks and incubate them after 70% confluence in serum free media for 48 hrs. Then use that supernatant for concentration first and once your CM is concentrated, use methanol-chloroform method for protein isolation. You will get a very good profile of proteins.
Hope it helps !
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs