Hello, I am trying to isolate exosomes from HT-29 cells by PEG precipitation followed by a 100,000Xg Ultracentrifugation wash. I am constantly getting low protein concentrations. I start with a culture of 10 ml. Can someone suggest me a way to increase the protein concentration? To visualize the exosome markers I need a minimum protein concentration of 20 ug to be loaded on the gel but I am merely getting 40-60 ug/ul with the Qubit protein concentration measurement kit.
Hello, I isolate extracellular vesicles by ultracentrifugation.
1. You can increase the number of cells from which you isolate exosomes.
2. Try to isolate proteins with e.g. RIPA buffer and sonication
Hi Klaudia
Thank you very much for your response. Would you kindly expand on your answers?
1. How can I increase the number of cells? So, basically, I have been using frozen HT29 cells, the initial number of which I do not know to begin with. I do a 10 ml culture and I am thinking about using 40 ml to be used with a PEG6000-NaCl mixture to increase the possibility of isolating exosomes. What method should I use to know the actual number of cells in my culture plates?
2. The papers I am following do not mention about a separate isolation method for proteins after exosome isolation. However, I read some research gate discussions where people have used RIPA buffer and sonication.
a. Should I use both at the same time or either one of them?
b. Do I need to use a protease inhibitor if I am using RIPA buffer?
c. Should I take the protein concentration after they are treated with the buffer+sonicator?
d. How do you typically go ahead with this RIPA buffer+sonication process?
Any suggestion will be highly appreciated!
Zubaida
hi.. are you sure that the total protein concentration you are getting is 40-60ug/ul...Then there will be enough samples to load on the gel?
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