I isolate my exosome through ultracentrifigation method from cell supernatant. In the last step I resuspend pellet in 200ul PBS 1X. I also characterize it with NTA to see size and number.
However I have problem with seeing band in WB. I use CD9 1:250 in 3% milk ON. I also have problem to see signal in dot blot while I could see in my positive control.
Did anyone has same problem?
Thanks for your help.
Hi,
I am also working on exosomes isolated from urine sample. Initially I also facing such problem. Then I standardized my western blot using CD63 (Santa Cruz) 1:500 and CD9 (Abcam) 1:500 in 2.5% SM at 37C for 2 hours.
Hi Zahra Motamed
If you're struggling to detect CD9 in exosomes isolated via ultracentrifugation, ensure you have a sufficient exosome yield by verifying concentration with NTA. Quantify protein concentration using methods like the BCA assay before loading. Use an appropriate lysis buffer with protease inhibitors to prevent degradation. Check the specificity of your CD9 antibody for exosomes and consider adjusting your blocking agent; sometimes 5% BSA is more effective than 3% milk. Ensure thorough washing steps, verify the efficiency of your secondary antibody and detection method, and always process and compare your samples similarly to your positive control. Using fresh samples or ensuring optimal storage conditions can also make a difference.
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