I am having problem in expressing my protein which has a m.cherry tag..does anybody here have ever faced this problem ?
Unfortunately, this is normal and you'll have to troubleshoot why. The first place I'd start is making sure that the construct was cloned correctly. Is it in frame with the tag? If it's a C-terminal tag, did you add a ribosome binding site? Assuming that's all good, you may want to verify that RNA is being transcribed as expression may be toxic. Without more details like the cell types and kind of expression system you're using, I can't be more specific.
first of all thanks for the response ...actually i am working on cytokine receptor ectodomains...what i did was i made a pcDna 3.1 construct which has receptor ectodomain with fluorescent tag(one gfp tag and for the other receptor ectodomain m cherry tag)eg for il-4 ...for il4 alpha---tagged to gfp and gamma chain to m.cherry...the constructs are perfectly fine(checked with seuencing)the problem is there is no problem with expression of gfp construct but the construct with m.cherry is some what cleaved and not detected in western blot..when i exchanged the the fluorescent dye(for gamma chain i changed with gfp)the expression worked....do you have any suggestions for alternative to m,cherry which is as efficient as gfp...transfection was done in hek293t cells by electroporation...please suggest me if u have any alternatives..thanx
mCherry and GFP are different proteins derived from completely different organisms. From your description it sounds like the mCherry fusion may be toxic whereas the GFP version is not. I'd try constructing another fusion with a different red fluorescent tag.
Hmm you are correct....i try to find some alternatives to m.cherry...if you hear from somewhere please let me know...nyways thanx a tonne
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