If you are using the same antibody for both IHC and Western blot, it is likely that you are detecting the protein found in the lower band in the IHC. You need to identify a more specific anti-TF antibody.

Just throwing this out there (it may be common knowledge), gene expression data need not necessarily correlate with protein levels. Transcript levels could be roughly equivalent, yet the TF protein could be stabilized in cancer cells but degraded in normal tissue. Moreover, the transcript could be translated at much higher levels in the tumor tissue but translation for that particular transcript could be inhibited or tightly regulated in the normal tissue. Additionally, your antibody might not pick up the TF in the normal tissue due to different post-translational modifications. As Adam mentioned, using another antibody would be highly beneficial to rule out specificity issues.

Hi there,

I agree with Erik, often there is no correlation between the mRNA expression and protein levels of protein of your interest in patient samples. The protein could be stabilized via many different mechanisms (mutation, de-ubiquitination, enhanced translation, PTMs that prevent Ub attachment etc). The lower band on the WB might represent the specific signal coming from a partially degraded protein (with present epitope recognized by Ab). Try and use several different Ab, with the epitopes located within different portions of your protein.

Good luck

Couple of points:

1- Protein and RNA expression correlate relatively poorly at the population level (40-60% only, depending on studies) so your protein of interest may indeed show a lack of correlation.  That being said, there is a limit to the lack of correlation and large changes to a given transcript will probably result in a matching change (directionwise at least, if not proportional) in protein abundance (everything else being equal...).

2- IHC and WB data cannot readily be compared as recognized epitopes may be quite distinct, reflecting quite distinct epitope presentation.  So the bands you are observing by WB may indeed represent protein(s) that are distinct from your IHC signals.  From personal experience the IHC field is plagued with results that make little sense (largely due to a lack of controls), so tread carefully there.  If you can use blocking peptides, good, but not enough.  If you have access to a KO tissue, much better.

Have you tried to look at cancer cell lines at all?  Is there a suitable model for your cancer type? If so, you may consider performing silencing experiments in a immunocytochemistry setting.  ICC is more similar to IHC than WB and  disappearance of your signal in ICC would add one more level of confidence in your data. 

Good luck!