Hello,
I've been trying to isolate exosomes from cell culture supernatant. I have spoken to other researchers who do exosome work and found that they don't use RIPA buffer to lyse the eVs when doing protein quantification (BCA) or western blots. They don't lyse the eVs at all. They claim that it degrades the eVs. However, in the literature, it seems common practice to use RIPA buffer or some type of lysis buffer. I have tried to find papers that support this claim but I haven't really found any. Just some papers comparing different lysis buffers.
Is the claim that RIPA or any lysis buffers isn't good for exosomes and characterizing them true? What to do instead?
Thank you for your time,
Britney
Hello Britney Hudson
Exosomes can be tougher to lyse than cells. You may have to use RIPA buffer at a higher concentration than it is intended to be used at (i.e., higher than 1X). But this may turn out to be much harsher than using the normal 1X RIPA lysis buffer. If you use a final concentration of say (5X) RIPA, it can improve lysis, but the disadvantage is that it may impact your protein of interest. It would be best if you try a range of RIPA concentrations on your samples that are not so precious and check for yourself which concentration works best for your protein of interest.
The claim that it degrades exosomes is right. Some researchers do not get good results with RIPA. Exosomes may be lysed using various other methods such as mechanical force or introduction of hypo/hypertonic solutions or detergent-containing buffers. If you do not wish to use RIPA buffer, I would recommend the use of the following lysis buffer.
Lysis Buffer recipe
Tris-HCl (pH 7.4): 25 mM
NaCl: 100 mM
Triton X-100: 1%
EDTA: 1mM
DTT: 1mM
NaVO4: 1 mM
β-Glycerol phosphate: 1 mM
Aprotinin: 1 ug/mL
Leupeptin: 1 ug/mL
For more information about the above lysis buffer and the protocol, you may want to refer to the article attached below.
https://star-protocols.cell.com/protocols/429
Best.
Hi !
I also working on exosome and I have the same question with Britney Hudson. I found a article protocol that they used Bolt 4X LDS Sample Buffer (Thermo Fisher Scientific) as Sample buffer and it had success extracting tetraspanins with LDS sample buffer alone. I think you can try to use this protocol.
https://arep.med.harvard.edu/pdf/Kowal_Church_2017.pdf
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