Hi everyone
I am getting a visible pellet of exosome. I added 100 ul PBS to dissolve it.after pipetting it was fully dissolved but after next day I found pellets in the bottom of 1.5 ml tube. Any suggestion what is that and what to do?
do I need to add more PBS or Warm it in 37c
please suggest.
Sandip
There may be protein contamination or else you need to user increase the PBS volume (with protease inhibitor). Hope it will work. And if it is still agglomerate then treat it properly according to your down stream use of that exo pellets.
Hello Sandip Kumar Mukherjee
I agree with Saikat Mukherjee . Sometimes, exosome pellet is difficult to dissolve because it may be containing contaminants in addition to the exosomes. It is essential to wash the exosome pellet with PBS at least twice to remove all the free protein remnants.
Best.
I agree with Saikat and Malcolm. It may be really hard to dissolve clumpy exosome pellets completely. I used to add PBS containing halt protease inhibitor to the exosome pellet, pipette and vortex it gently, and then put it on a shaker at least 2 hours (or overnight) at +4 centigrade degrees. I think, increasing the volume of PBS wouldn't be a problem.
Hi,
If you are getting a visible pellet of exosome by end of exosome isolation/purification process, then adding 100 ul PBS is less and using 1.5 ml tube is also not suitable as exosome attach to the surface of the tube, resulting in loss of exosome. So I will recommend adding 500-600 ul PBS and then storing it in 1.5 ml tube. And if you are doing the Exo-isolation process correctly, then most probably the dissolving problem won't happen while passing the media through 0.2um filter before Exo isolation and washing the isolated Exo with PBS could be more helpul.
Warming at 37 degrees won't be a good practice.
Good luck
I used to wash the exosomes 1-2 times with ice cold PBS after a 0.2 µm sterile filtration, just to avoid any proteins contamination!!!
A visible pellet indicate a huge amount of exosomes, which can form aggregates very fast. I would recommend to increase the PBS volume to 500 µl with gentle and persistence agitation (in case you want to use the functional exosomes).
Good luck
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