I am performing splicing of dystrophin in cancer cells. In one cell line, no conventional dystrophin transcript was successfully amplified by PCR. We supposed that this cell line habor multi intron retentions and consequently, no dystrophin protein will be identified.
To our greatest dismay, western blotting as well as immunostaining indicated dystrophin expression.
Is it possible to identify protein and not mRNA of a particular gene? Are there other examples of such phenomenon?
Thank you
The specificity of the signals concerning protein expression should be confirmed by using RNAi.
Even if molecular size of your signals on SDS-PAGE seems to be correct, it is possible that the signals are derived from non-specific target protein.
Any antibodies are likely to show somewhat cross-reactivities against other proteins besides specific target.
How about performing immunoprecipitation to check whether the protein is concentrated or not.
I am sorry. My favorite field is DNA dynamics; replication, recombination and repair.
If you demonstrate that a protein is translated directly from DNA then a Nobel Prize is not far away.
mRNA levels do not always correlate with the corresponding protein levels, especially with time.
Maybe the mRNA you want to detect is especially unstable.
Another factor worth checking would be the specificity of your PCR primers.
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