Hi all, I've been isolating exosomes from cell culture media using ultrafiltration (protocol recommended by Merck). So far, I'm getting good number of exosomes. However, I'm having a bit of trouble in extracting proteins from exosomes.
The exosome samples that I collect after ultrafiltration are in the form of solution (they do not form pellet). I add RIPA buffer to these samples (60 ul sample +100 ul RIPA), keep it at -20C overnight and then centrifuge it at 4C for 20 mins. Now since there is no pellet formation, I'm not sure I'm doing it right.
Furthermore, when I run Western blot with these protein samples, the lanes have sort of a smear, which makes me think there's an issue with the protein extraction.
Any help will be highly appreciated. Thanks
Dear Shalini,
The buffer you are using should not be the problem. Maybe the lysis protocol is a bigger issue. You might wanna try lysis at 4ºC for shorter time in a rotating platform (30 min to 1h it's already sufficient). If you want to improve the lysis, pulse vortexing before lysis will help you resuspend the EVs in the lysis buffer.
It is normal to not observe a pellet after centrifugation when working with lysed EVs... That should not be a problem!
Hope this helps you with the protein extraction.
Good luck and best regards,
Julia
We use the following Lysis Buffer: 1% Triton X‐100, 50mM HEPES, pH 7.4, 150mM NaCl, 1.5mM MgCl2,
1mM EGTA, 100mM NaF, 10mM Na pyrophosphate, 1mM Na3VO4, 10% glycerol,
containing freshly added protease and phosphatase inhibitors from Roche Applied
Science Cat. # 05056489001 and 04906837001, respectively.
You can also add sample bufferx5 to your exosome pellet and boil without lysis. The unusual running could be related to high salt concentration, PH, protein concentration etc. We usually load 50 microgram total protein.
Hi Shalini Paul I saw you centrifuged sample after lysis in 20 min but I am not sure about the speed, maybe you should try to use high speed of centrifugation to pull down the protein.
Hi Shalini Paul,
We are currently conducting a good study on the exosome. We've been isolating exosomes from 100 ml cell culture media using ultrasentrifuge. We can see exosome pellets after ultrasentrifuge. We add 100ul RIPA lysis buffer (containing 1% 10 mM PMSF and 1% phosphatase inhibitor cocktail) to these pellett. After the samples are freeze-thaw 3 times in liquid nitrogen, we keep them on ice for 5 minutes and then it centrifuge at 4ºC 13000 RPM for 10 minutes. Supernatant is transferred to clean tubes.
If you want to see more concentrated protein, you can dilute samples the using 10x Laemmli Sample Buffer (EcoTech Biotechnology, Erzurum, Turkey) and then ıt boil for 5 minutes at 100ºC. Therefore, it will be sufficient to load 20ug total protein.
thank you all for your kind answers..I shall try them out and see how it goes
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