Hi everyone,
I am starting to work with extracellular vesicles from mesenchymal stem cells cultured with Messencult ACF-Plus medium. The technique used for their isolation is ultracentrifugation.
First, we want to characterize them trough western blot. I am having some trouble with protein isolation and western blotting. I’ve tried RIPA buffer 1X and 5X. With both RIPA concentrations, protein is quantified trough BCA assay but, after electrophoresis and protein transfer, the Ponceau S staining looks as shown in the attached image. Though it looks like EVs proteins begin to separate, eventually EVs proteins remain stuck at 60-50kDa. This only happens with EV protein because cell lysate runs without any problem.
Electrophoresis conditions are: 30 ug protein/well, 10% acrylamide gel, non-reducing conditions. Samples are previously denaturalized 90ºC for 5 minutes.
Transfer conditions are: 400mA for 2 hours at 4ºC, nitrocellulose membrane.
Can someone help me? What can I change to improve EV protein isolation and western blotting?
Thanks :)
Hi Paulo!
It seems that the molecular weight of the extracellular vesicles (EVs) is around 50 kDa, which aligns with the Ponceau staining at the same size. However, if you're not observing multiple bands in the Ponceau staining of the EVs, it doesn't necessarily indicate that the proteins are concentrated near the 50-60 kDa range. In some cases, even pure samples may not show multiple bands, and I've found that certain lysates can also exhibit a similar lack of complexity after Ponceau staining. To better verify your results, I recommend processing your membrane with EV-specific markers such as CD63 and CD81. Additionally, it appears that there may be some overloading in your lysate and purified EV protein samples. I suggest loading approximately 10 µg of the lysate and 1-2 µg of the purified EV sample for optimal results. Please share your findings, and good luck!
Hi, I’m having the same problem. Were you able to solve it? I’ve repeated the experiment several times, but I keep seeing the same results with my EVs, just as you described.
Hi, I’m having the same problem. Were you able to solve it? I’ve repeated the experiment several times, but I keep seeing the same results with my EVs, just as you described.
I'm currently running a new experiment with these recommendations.
Gaurav Chhetri thank you so much for the advice.
I'll keep you update with the results :)
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