Hi,
I'm working on exosomes isolated from cell supernatant of cultured cells. Briefly, I accumulate 100 - 200 ml of supernatant and process it until a concentration in a final volume of 50µL.
Then I want to be sure of the presence of exosomal markers but I have so trouble with western blotting and there is a lot of discrepancy in literature about this subject.
1. Should I lysate my exosomes in RIPA buffer or should I load them directly on my gel ?
2. Should I use DTT in my laemmli buffer to load my sample ?
3. Is there another trick to have beautiful exosome western blot ?
Thank you in advance
Delphine
1. Yes, you should lyse your exosomal samples with RIPA buffer or lysis buffer of your choice.
2. If you are blotting for CD63, CD81, CD9 marker, you should avoid reducing agents like DTT or BME because disulfide bonds are required for antibody to recognize epitope of these tetraspanine proteins.
3. Unfortunately, the only trick is paying attention on what you are doing and always including appropriate controls. ISEV recommends negative controls for intracellular contaminants such as GRP94, calnexin for ER, GM130 for Golgi, CYC1 for mitochondria. Good luck :)
1. Yes, you should lyse your exosomal samples with RIPA buffer or lysis buffer of your choice.
2. If you are blotting for CD63, CD81, CD9 marker, you should avoid reducing agents like DTT or BME because disulfide bonds are required for antibody to recognize epitope of these tetraspanine proteins.
3. Unfortunately, the only trick is paying attention on what you are doing and always including appropriate controls. ISEV recommends negative controls for intracellular contaminants such as GRP94, calnexin for ER, GM130 for Golgi, CYC1 for mitochondria. Good luck :)
I totally agree with Nghiem Le. Try what he suggested, you will get good results.
Good luck.
I work with exosomes. I lyse my samples with RIPA buffer and you can use other commercial lysis buffers. I did not face any problems while blotting for CD63, CD81, and CD9 exosomal markers. Question is did you check the size and size distribution of exosomes? What method you are using to isolate them? Is there FBS in your media? Are you using appropriate negative controls? Is the amount sufficient that you are running in a gel? You can also run a cell lysate just to check whether your antibodies are fine.
Hello Delphine,
If I may add to what has been already said - CD63 is often glycosylated, which can be an issue for good WB detection. You may want to consider treating your sample with PNGase F for example.
We run our westerns under denaturating conditions and have no problem. A lot depends on the antibodies that you use.
Good luck!
Hello,
thank you for your advices. I would like to know, what volume do you use to lyse exosomal samples with RIPA buffer, 1:1 dilution or something else? I concentrate samples with Speedvac to get appropriate volume for western blot, would you recommend to add RIPA buffer before Speedvac or after? Do you use different method of concentrating samples?
Thank you.
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