Greetings, I am trying to detect transient protein-protein interactions in tissue lysates from mouse skeletal muscle. I have tried co-IP, and even though I am able to get a good IP of my protein, I am not recovering the interacting proteins. I was thinking of using a chemical crosslinker (such as DSS) to stabilize the protein interactions before doing a co-IP. Did anyone try that in a tissue lysate and could share any advice/reference/protocol. Thanks so much in advance!
Hey Dmitri,
have you tried the cross linking of the lysates? I have the same problem with my Co-IPs.
Thanks in advance,
David
Hey David Khangholi ,
I was able to get it to work without the crosslinking. The idea was to greatly increase the amount of protein used for IP. I had to spin down the tissue lysates at high speed (100K g, 1h) before starting the IP. By doing so you remove the inpuriteis in the tissue lysates and are able to use more protein (3.5 mg/sample) for IP, while reducing the background. It worked well for me and and the results were published in https://diabetes.diabetesjournals.org/content/67/7/1272.long
See the method section and the figure legend for Figure 6.
Hope it helps.
Good luck,
Dmitri.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue