Hi All, I need to perform double transfection in raw 264.7 with mouse p50-flag tag and his tag to know p50:p50 homodimer formation? I need to know is position of tag going to affect transfection?
What is the criteria for choosing tags and their respective position in vector?
I need to know is their any other way out apart from double transfection to know presence of formation of homodimer.
We have used Ra 264.7 macrophages in the past. Although they may be difficult to transfect, the transfection shouldn't be affected by the position of an epitope-tag.
We normally attach an epitope tag at the N-terminus. If the N-terminus is required for post-translational modification (N-myristoylation) or contains a cleavable signal sequence (for example, class B G-protein coupled receptors), then we add an epitope-tag at the C-terminus.
Co-transfecting p50-Flag tag and p50-His tag expressing plasmids is the easiest way to study homo-dimerisation. Yes, co-transfection may effect the levels of expression of p50 with different tags. However, it shouldn't be a problem if you are planning to study homo-dimerisation qualitatively.
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