Hello all,

I am dealing with a transmembrane protein and trying to create constructs with different domains of it. The one I am particularly interested in is the intracellular domain (8.5 Kd) containing a predicted NLS.

One expression vectors containing the ICD was already made by a post doc in the lab, I just PCR amplified the gene with and without its nls domain and inserted it into a untagged(prk5) and c-terminally flag tagged vector (both contains CMV promoter) so that I can get two  truncated version of the protein and confirm its localization.

After checking and rechecking the sequence of the clones, transfected them in HEK cells (which do not express that protein), prepared post nuclear cell lysate after 48 hours and run a 4-12% gradient gel.

I almost got no expression ( got very faint, sad smiley looking bands) from the constructs I created but got nice bands (at 9 kd) from the constructs made by the other person, though the vector backbone, transfection procedure, cell handling conditions, western blot conditions all are same. I have done ICC and also got positive staining for my expression constructs (although background was high).

I again checked the maxipreped DNA sequence, done digestion and got desired product but no expression at the protein level at least in western blot.

Now, I am thinking to check at the RNA level by doing rt-pcr, but can anyone of you tell me what could go wrong or any suggestions/ explanation regarding this situation would be very helpful.