Protein cloned in pRK-5 Myc and pcDNA3.1 Myc-His Tag not showing up - I need help for a transient expression system (Feb/18/2010 )

Hello every body
I need some help to transiently express a protein. I inserted from a cDNA derived from RNA of a patient. I sequenced the product and it is fine. I do not have frameshift and I do not have stop codons in the coding region. I cloned this product for the first time in a pcDNA3.1 His-Myc tagged at the C terminal and gave it to the person who will do the experiment. He made a transfection with lipofectamine and a western and could not see any product. The protein is 1232 aminoacid long. The detection was done with an anti-Myc antibody after 24 hrs transfection. I guess he made a blot with an anti-His too and the result was the same. So he told me could be a problem of the vector...he hasd already had problems with this vector, so he suggested me to try again with another expression vector that in his hands is working more than fine. So I made the constructs once again this time in a pRK-5 vector with a Myc tag at the N terminal. Once again sequenced the construct and every thing was in frame. Gave back to him the construct and again lipofectamine transfection, western and no differences between cells not treated- 24h and 48 hrs of expression.

Now should I try yet another vector? What can be wrong? The cells we have to transfect are epithelial cells A539 (if I'm not mistaken). We tried with the first vector to transfect the HEK239 with a protocol optimized for another vector, but no luck either....no bands at all......

So 2 vectors 2 fails?

Can you suggest any other vector or anything that could be improved to make things work?

Thanks for the help in advance

Micky

I know its might be a stupid suggestion, but do you have a positive control for the myc antibody. Also i guess you have verified that myc sequence is in frame with rest of the cDNA, like no stop between them and other weird stuff.

cheers

scolix on Feb 20 2010, 11:06 AM said:

Well the person who is doing the WB told me that he has a protocol which is always working fine for the myc antibody. I checked with sequence and the myc tag is in frame and no stop codons detected, bot for the 3' and the 5' tag

I agree the protocol works for myc, but in the westerns is there any positive control for myc. Maybe the antibody is not working as it used to. could have got degraded. If there is any old sample with myc, run in on the same WB to make sure that the WB is working.

How is the transfection efficiency in cells? I would wait for atleast 48 hrs before making lysates from the transfected cells.

good luck