IXA

Hi everyone,

I've been following this forum for a while (and it has always proved useful) but this is my first post on the site.

I've been having numerous problems cloning and expressing a gene of interest in a pCMV-Tag3a mammalian expression vector for quite some time now.

I've had my construct sequenced and the N-terminal myc tag is in frame with my protein of interest.

However, upon transient transfection into HEK-293Ts, I am unable to detect expression the construct through Western blotting. I use a pCMV-Tag3a mammalian expression vector containing another protein (generated by someone else in the lab a number of years ago) as a positive control and can detect this through Western blotting with no problems. So I do not feel I have an obvious transfection issue.

I'm not quite sure where to go from here...

Appreciate any advice or help.

Thank you.