Any suggestions on why tag is not working in western blot?

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Tanmoyita Nayak...
Tanmoyita Nayak Nayak's picture
Any suggestions on why tag is not working in western blot?

 I made a clone with my insert (230bp) and vector (5kb) which is c-terminally flag tagged. After making the constructs I have checked by sequencing twice! (it`s sure that there is not any stop codon before the flag tag). However, my clones are not working when I run a western blot with them, they are giving correct band when I treat them with the gene-specific antibody but no bands when its treated with the anti-flag antibody (sigma , mouse monoclonal).
I have already tried different batches of antibody with my lab and from some other lab as well.
I have also done immunoflouroscence imaging with flag antibody, though there is high background but still the cells were stained.
I work with whole cell lysate and blot for 2 hours, 300mA, PVDF membrane.
I am not sure what can I do to make it work in western blot. If you experts can help me regarding this, I would be really thankful.

Chin Fen Teo
Chin Fen Teo's picture
Hi tanmiyitank,

Hi tanmiyitank,

I suspect your problem rises from invalid western condition- You can try to perform a serial dilution of your primary antibody ranging from 1/5000 to 1/20000 dilution to first optimize the best condition of the anti-flag antibody used in your western. The other thing you might want to consider is to get a control flag-tagged proteins that others have succesfully detect with western blot to serve as your positive control.

Good luck.