I cloned a segment of my gene of interest in PCDNA 3.1. I checked the sequence by sequencing and everything was fine. Then, I transfected my construct in HEK293 cells. After transfection I could not detect any band by western blot using anti V5 antibody. The vector is tagged with V5 tag. But when I transfected control plasmid which was also tagged with V5 epitope using same conditions for transfection it detected band by western blot. Same amount of protein was used for the blot. My protein was approximately 33kda which I am expecting.
Since my protein was a small part of a big protein is it possible that it gets degraded during transfection.
Since your antibody and the transfection seems to work, I would take a look onto your protein. It could be degraded, which you test by using a proteasome inhibitor for some time before you harvest your cells. If your problem is breakdown, you should get a band then. It is also possible that the protein is not expressed very well (is your protein toxic for the cells?) or that the vector or the mRNA is silenced by the cells. I guess you sequenced the vector to make sure it is correct?
If you vector has been constructed correctly there are really only three possibilities:
1) transfection was unsuccessful - do co-transfection of both vector and control-vector or use any other proteins on your vector which may be expressed as a control
2) Low levels of protein present in cells. This could be caused by protein degradation or low expression levels of your gene - add proteasome inhibitor as christian suggests above or do northern blot or qPCR to check mRNA levels. Alternatively, try different cell line.
3) your tag is inaccessible to primary antibody - express tagged protein in E. coli and purify, then do western on a titration of your protein to test how low amounts you can detect using your primary antibody.
Good luck
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