I am using Sigma rabbit anti-FLAG antibody for the detection of AAV-driven expression of a 3×FLAG-tagged protein in mouse brain however signal occurs nearly everywhere in the whole brain except the cells should be express the protein. Using this antibody for Western blot very weak unexpected bands occurred which were not seen by M2 anti-FLAG. Does anyone have experience in the detection of FLAG/3×FLAG epitope in brain sections? Is it possible that special conditions are needed or different manufacturer’s antibody should be used for this application?
From my experience, FLAG antibody never worked reliably on IHC of FFPE brain sections. It works well for Western but not for IHC. For IHC purposes we have used HA tagged constructs, which seem to have much cleaner and reproducible results. Best of luck.
Thank you for answering, Pavel. Did you try to change conditions which resulted improved staining (e.g. modified fixation protocol, buffers etc.)?
Yes we tried many conditions with no significant improvement. After this experience I would not trust FLAG for detecting transgene expression in the brain.
Hello Peter, have you got new results?. I must say that we have had a similar issue. Our AB is Monoclonal ANTI-FLAG® M2, Clone M2 from Sigma. I would like to talk a little bit more about this. This is my email. [email protected]. Best!
Hi José, Yes, but only bad results, the M2 could be the best aFLAG antibody, but the problem is that we are working on mouse brain in a region where the BBB is incomplete. The biotin-conjugated M2 could be the next option. Peter
Immunohistochemical Detection of FLAG-Tagged Endogenous Proteins in Knock-In Mice. J Histochem Cytochem. 2015 Apr; 63(4): 244–255. this paper is talking about this.
it seems a common problem.
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