I introduced V5 tag by CRISPR to a nuclear protein with low level of expression and I detect dotty staining just a bit more intense than a background. I didn´t want to use GFP because of the size but now I´m not sure it was wise decision. I placed the tag on N-term of my protein because the C-term has important domains for binding and nothing is known about the N-term. I need the tag to follow the protein distribution in the nucleus and also for the IP/CHIP. I´m using Abcam mouse antibody against V5, it works very well for the overexpressed proteins.
We use these epitope tags called "spaghetti monsters" to see proteins of extremely low expression. The spaghetti monster protein is a modified GFP that has 10-13 epitope tags and the protein is unable to fold, thus it forms a long spaghetti-like structure in vivo. The GFP is not active (so it doesn't fluoresce) and the monsters come in HA, myc, V5 and other tags. We use them in Toxoplasma gondii to see lowly expressed proteins and it works extremely well. Anti-GFP doesn't recognize it so you can still use GFP in cells too.
http://www.ncbi.nlm.nih.gov/pubmed/25915120
https://www.addgene.org/browse/article/8960/
Hope this helps!
Hi Andrew,
Thanks for your suggestion. I´m reading the paper now. I might have some questions to you after...
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression