I used 100ml of secretome to isolate the exosomes through ultracentrifugation. Then, I tried to lyse with 1X RIPA or 5%SDS along with 10s sonication. Later, added loading buffer of SDS-PAGE, boiled at 95℃ for 5 mins. I did the NTA with the same isolation method and volume; the concentration of exosomes was quite good. However, I am not getting any results on my WB.
Haidar Fayoud
If you are sure of the isolation, this means that the lysis was not successful. Try increasing the sonication amplitude (adjust it to ~30%)- make sur to work on ice, otherwise your proteins will be degraded- and extending the boiling time to 10 minutes.
0 votes
0 thanks
Louis-Charles Béland
How much protein are you loading on your gel? Try to at least put 10-15µg. Do you see bands with Ponceau ou a similar method?
0 votes
0 thanks
- Badges
- Science topic
- Similar topics
- Computer Science
- Research Topics
More Mohil Mishra's questions See All
Similar questions and discussions