I am working on exosomes as a part of master’s thesis and am facing one major problem regarding the characterization of the exosomes using the western blotting technique. I have isolated the exosomes from human umbilical cord-derived MSCs which were cultured in α-MEM complete media and after they attained confluency the media was changed to serum-free to avoid the precipitation of any extracellular vesicles from serum itself. After 48HRS of treatment with serum-free media, the exosomes were isolated by Total exosome isolation kit (by INVITROGEN) following the manufacturer’s protocol. The protein content was measured to be 536.27µg/ml. The exosomes were thereby suspended in suspension buffer ( for 10ml media – 500µl suspension buffer) and kept at -20 degree Celsius. The protein content was measured to be 536.93µg/ml by BCA assay. The 10% SDS gel was prepared and the samples (40µl sample with 20µl loading dye ) heat treated at 95 degree Celsius for 5 to 10 mins. After running the gel at 100V, the gel was transferred manually to the transfer apparatus with PVDF membrane for 1 hr at 18 to 19 mA current for 1 hour. Blocking was done for 1 hour with 5% non-fat milk in TBST. I have done the blocking for two and three hours respectively. But it didn’t yield any result. After blocking washing with TBST for 3 times 10 minutes each was done and thereafter it was incubated with primary antibody – anti-human CD9,63,81 (I have tried with 1:1000 and 1:2000 dilution.10µl for 10 ml and also 10µl for 20 ml BSA solution) on a gel rocker overnight at 18 degree Celsius. The next day, washing has been done 3 times for 10 mins each and incubated with secondary antibody-anti rabbit HRP IgG (3.3µl for 10 ml and 6.6µl for 20 ml TBST & 5% non-fat milk solution) for 2 hrs. Thereafter washing was repeated 3 times 10 minutes each. The chemiluminescence substrate was added (A: B= 1:1) 1ml and incubated for 5 minutes after which it was viewed under a ChemiDoc imager. In spite of following the protocol and trying it several times, I didn’t see any band other than the granny background which is mostly of the non-specific binding supposedly. Can you please suggest me some alternative which you think might work or if there is any problem with the protocol I am following?
Hi,
It should be straight forward with kit.
Here is protocol, methods from colleague of mine.
http://www.pnas.org/content/113/8/E968
It works pretty well. Another one is this
https://www.nature.com/articles/ncomms8506
Use standard antibody for Western blot.
Good luck.
Anand
It would be a good idea to assess the quality of exosomes after isolation from your media by a sizing profile. Usually exosomes are collected in buffer, and they will increase in size some secondary to oncotic forces. It is likely, even though you changed to media without serum, that you have a lot of albumin and other serum proteins still present in the cell culture dish, and this will give you high protein readings. A sizing profile will help you determine if you are getting any exosomes and if so, you will likely need to work at removing remaining serum proteins as first step of exosome isolation. This can be done with size exclusion column chromatography and is not difficult.
The problem can be with transfer or detection. I will at the beginning check the transfer.
I usually make a semi dry transfer (I deduct that this transfer did you use also) through 1 h but with at least 150mA (membrane has 30 cm2) - recommendation is to use 1 – 1,5 mA for each cm2 of membrane, but for bigger protein the higher current you use the better transfer you have. PVDF membrane has to also be activated in pure methanol and balanced with blotting buffer (with 20% methanol).
To check if transfer was ok you can try to use Ponceau S solution it is reversible staining, you will see if proteins are present on the membrane and later you can wash the membrane and follow standard procedure. There is also easier way to check - just stain your membrane with coomassie blue, but you cannot use this membrane to immuno detection anymore.
If you have the problem with detection maybe your Ab are wrong, to much dilluted or unspecific or the chemiluminescence substrate is not working - use another one (borrow form somebody a little just to check).
Good luck!
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