I will do some sample yield tests because I will work with exosomes of patient urine samples.
For mass spectrometry
I like to know:
1) How you are preparing the urine sample (lysis buffer?) ?
2)How you are Isolating exosomes (column or ultracentrifuge?)
3) How you are Quantification (Nanovue or BSA?)
4) How you digestion for mass spectrometry (trypsin or other enzyme) ?
It is a way to improve exosomes isolation; you can follow optimized ultracentrifugation approach to obtain highly reproducible isolation. So, using a protease inhibitors (inhabit from exosomes structure damage) and DTT (disperse trapped exosomes from pellet) at initial step, exosomes will be conserved abundantly. Consider that 50 ml of sample is recommended. Following 6 steps centrifuging you can reach a sufficient volume of exosomes to evaluate RNA, proteins and etc.
Great answer Jafar Rezaie!
Many researchers ask me about commercial lysis buffer buffers. Have you used it on your own?
My protocol is the same as yours. I've never used the commercial lysis cap.
Thank you
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