Dear all,
I run into a extremely trouble. And now I turn to your help. I have constructed flag-biotin tagged PRC2(EED, EZH2, Jarid2, MTF2) knock in cell line by HA to one allele of protein.I take the positive flag-biotin-EED,EZH2,Jarid2 cell line by PCR screening and southern blot. Here is my trouble: I excise the neo gene by flpase. And then I also detect the cell line by PCR for 5'end and 3'end and southern blot. Question is all of confirmation is right in molecular level. But in protein expresion level, I can use flag beads and streptavidin beads to Co-IP interaction protein. For example, FLAG-biotin-EED, I can use flag tag CO-IP.The suz12 signal can be detected. But I can not detect FLAG tag itself by anti-FLAG. And I use biotin tag of EED to perform CO-IP. The Suz12 signal also can be detected and the biotin tag signal can be detected by streptavidin-HRP as well. Why the FLAG tag can not be detected by anti-FLAG but can be recongnized by FLAG M2 beads??? Who can tell me the reasons? It will be great appreciation! Thank you!
Here is my analysis, I think flag tag can be recongnized by FLAG M2 beads by CO-IP such as in flag-biotin-EED cell line. I can detect EZH2,SUZ12. It shows that the flag tag is expressed in my flag-biotin-PRC2 cell line. In SA-IP sample, I detect the flag tag. But no FLAG tag signal by incubating with Anti-flag (sigma,mouse). It shows that FLAG tag may be not expressed.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology