I'm using the Total Exosome Isolation Kit (from cell culture media) by Invitrogen and comparing it to ultracentrifugation.
However, I can't seem to get any bands in the TEI kit during Western. I'm probing for CD63, MHC-II, and Hsp70. I've gotten bands from the UC method for Hsp70 but for my purposes that's not ideal.
I've read that exosomes are damaged during UC and that makes me ask: Do exosomes from the TEI kit need to be lysed before SDS-PAGE? I know these antibodies work, the bands appear in lysates at the proper weight. I've heard RIPA is a good buffer. How much would I add? 100ul?
The transfer seems to work fine using an iBlot. Blocking is 5% milk in TBST. The cells I'm using are MDCK.
UPDATE:
Thanks for your responses.
As a test, I created five separate preparations of exosomes. Each has a different method of lysis under normal and our typical experimental condition. 20ug of protein was loaded and run under reducing conditions. None of the exosomes isolated by TEI yielded any bands in CD63 no matter how long I exposed for. The lysates, though, were readily visible and could be picked up almost immediately, so it isn't a matter of concentration or antibody. These exosomes were never thawed or even frozen and were isolated per the manufacturer's instructions, so any kind of degradation is unlikely as well since they were put into SDS-PAGE the same day. There were no exosomes in the media beforehand, either. While it did yield an impressively-large pellet, I am not sure what the kit is isolating since none of the traditional exosome markers show up.
I ran it under EM as well. There are exosome-like structures. Many of them! But no protein markers, which makes me think these are maybe something else in the 25.6nm range.
As a recheck, I stripped and reprobed the membrane for HLADR and Hsp70 as well. Visible bands in the lysates. No bands in the exosomes. I used the same method of lysis for the cells as I did for the exosomes, both in RIPA with protease inhibitor cocktail present.
Isolation of any membrane bound material such as exosomes need lysis using detergent containing buffers before Western. Triton-X100 and NP40 containing buffer should work.
You would certainly want to lyse. However, others have reported protein detection difficulties after using precipitation-based kits. These kits rely on "salting out," often with polyethylene glycol, and are not specific for exosomes or other extracellular vesicles. In most cases, they will provide the least pure vesicles of the various methods currently in use, although they have their uses, such as concentrating everything in a dilute sample before other methods are applied. Ultracentrifugation is still the most widely used method in the field. For many experimental purposes, any changes caused by UC are irrelevant, but there is currently a shift towards gentler methods ranging from size exclusion chromatography and field flow fractionation.
I our case, we do not lyse our exosome prior SDS-PAGE. We just add Laemmli buffer, heat the sample for 5' at 95C and we are able to detect CD63, CD9 and others proteins that are supposed to be inside the exosome.
Dear Colin, I've isolated exosomes from different cancer cell lines by using the same kit (TEI) and resuspended them in 200 uL as suggested in the protocol. I've observed vesicles from 25 and 45 nm in diameter by TEM. Moreover, I've quantified proteins without lysing them before, I got from 2,5 and 3,6 mg/mL. I'm going to run a WB tomorrow without lysing exosomes in RIPA buffer before because I think that incubation in Laemmli Buffer before performing the WB is sufficient to lysate exosomes. I'll tell you about my results.
Best regards
FM
I cant dissolve isolated exosomes in 5% SDS for protein analyses and someone can suggest any ideas ?
I've used the Thermofisher precipitation kit for plasma. The only major contaminants I get are immunoglobulins that show up prominently as heavy and light chains, but I avoid that by not using reducing agents in my Laemmli (it's recommended to not use reducing agents because it can affect your bands for tetraspannins like CD9, 63).
Also, I found that when heating the samples prior to running in my gels, 10 minutes @ 70C worked much better than 5 min 90-100C. Don't know why but the bands just come out crisper. I've never used RIPA (nor protease/phosphatase inhibitors) on my exosomes.
Which ECL do you use in order to detect your exosome bands in the blot?
I use ECL 2Western or SuperSignal WestFemto (Thermo) but I am not very happy.
How many exosome ug do you load in your experiment in order to detect exosomes band?
Thank you
So, I don't really use ECL anymore. After the bad batch of it that had me tearing my hair out for weeks, I'm averse to the stuff. When I did use it, I used LiCor's WesternSure or Thermo's version of it. Both seemed to operate around the same sensitivity. When I got really desperate, I tried WesternSure Premium. It does seem to be better, but it costs an arm and a leg.
I've actually converted straight to near-infrared fluorescent Westerns. I find these are better for exosome research since the sensitivity is higher, there's no sprint to the darkroom, and I can probe for two antibodies simultaneously versus one at a time in ECL. This saves me a ton of time since I only have to strip two times.
For loading, I don't do any protein assays beforehand. In the course of ultracentrifugation, proteins tend to copellet, leading to bizarrely high reads with Bradford/DC. Even after several wash steps, all you really do is wind up losing exosomes since if the protein pelleted the first time, it'll pellet the tenth. Anything short of SEC or DG can't really remove that stuff. Instead, I simply load a predetermined volume of UC exosomes. From there, I get a particle count and can say I loaded X exosomes.
What kind of lysate do you use for your positive control? Regarding lysis buffer I normally use RIPA but I find a lot of albumin in my exosomes samples.
We just solubilize the exosomes in sample buffer and subjecte them to SDS-PAGE and Western blotting.
Hi, did anyone tried TCA precipitation for exosomes before SDS page ? In my case protein concentration as determined by BCA came too less , so i tried precipitating protein (vol. corresponding pto 30ug) by TCA and then resuspend the protein pellet in 2x laemelli buffer to load into SDS PAGE .. but so far I couldnt see any bands , i am probing for TSG101. Any suggetsions will be helpful .
Thanks
Colin Germer this paper might help you. Hashemi et al 2018 https://www.nature.com/articles/s41598-018-22142-x
Laemmli buffer did the trick for me, when running a SDS-Gel from EVs.
Dear all,
Is there anyway to denature EVs like heating it so that denatured Evs can be used as Vehicle in animal studies?
With regards,
Raghavendra
The visibility of some markers by blotting is highly dependent on the cell of origin used for isolation. For example, you'll likely see Alix in MDA-MB-231 cells while they are enriched in U87-MG or HEK-293T cells. TSG101 is abundant in 231 cells but I have had difficulty probing it in U87 or the SNU profiles.
Since you saw vesicle-like structures under EM, you might want to blot the exosome fractions with GM130 or negative markers just to confirm the absence of such in your EV blot, if there is GM130 expression, then likely what you saw under EM are apoptotic EVs and not exosomes themselves.
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