Hello,
I isolated exosomes from DHL-16 (DLBCL) cell line, using traditional ultracentrifugation process and validated its size using Nanosight. The size obtained was within the range of typical size of exosomes, but when I went on to perform validation using a western blot, I was not able to detect any band using TSG101. Is there any other marker that would work best for DLBCL cell lines? Any inputs or suggestions would be very helpful!
Thanks,
Hi,
as you have "Process Validation" listed I have to ask, if you are sure that your Western worked properly. To have a step by step monitoring of your whole exosome purification procedure you could load a sample from each seperation step on the gel/blot. With the help of a protein labeling (SPL Western Blot technology) you can then visualize all proteins, track them during the whole WB process and then detect your marker(s). This way you can be sure that you have not lost too much protein on the way and you can see differences in the protein patterns of your fractions. Hopefully you see how the marker protein(s) fade in one fraction and increase in your exosom fraction. And you can be sure that your detection is working.
I tried to purify exosomes in my PhD too. I used HSPA 8. I wanted to analyse them in a 2D-DIGE Gel but never got enough protein to actually get a preparative gel. I hope your exosomes will be more cooperative than mine :)
I hope you will find a good maker,
Kristin
Dr. Kristin,
Thanks for your suggestion. The marker that I am using is just to validate exosomes,and I see a clear banding for all the positive controls that I have used. Yes,may be concentration was an issue. I will try replicating with a different marker and let you know how it goes.
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