It was our experience that the traditional two-step Western is not good for all proteins, especially those trace ones.  We simply conjugated our primary antibodies with HRP and use a one-step Western for enhance sensitivity and specificity (keep in mind that the secondary Ab-HRP cross-recognizes IgG of other species).  Check out our paper for details: PMID: 25900303. 

Well, it's kind of hard to see because the picture is small, but it seems to me that what you actually have there is two bands (in the extremes of the smear), and between those two bands, the "ovelaping" of both bands' limits resulting in a well stained blur. 

Besides, even if that is a smear, and I'm seein wrong, plenty of phenomena add up to smeared bands, from gel quality, run parameters, incomplete denaturation, complex protein interactions both prior to run and during run, remaining post-translational modifications of the protein.

In SDS-PAGE, molecular weight-depending migration can be very usel but very deceiving, because it only trully happens for perfectly denatured, perfectly sugar-free protein. You can go on believing that your protein iis there - which is perfectly reasonable - or you can repeat the gel, applying a more intense denaturing procedure prior to run. (By the way, how did you denature your protein?)

Best regards

The protein was denatured with heating at 95, SDS and TCEP as a reducing agent. Western Blotting that followed with an antibody against the actual protein itself (not tags) seemed to verify that it was there ( i say seemed because it ain't getting the clarity award either)

Before adding the protein in the gel, the affinity tags were cut by enterokinase (which might be the clearer band up above)

Interesting hypothesis about two different bands, thank you.

Well, denaturation seemed to be OK. If it was a recombinant protein containing tags, that would explain the existence of 2 bands... How big was the tag?

Hmm, the combined weight of all the tags, 6x His, Thioredoxin and S-tag was at around 17 kDa. I don't think it would fall down there?

Maybe proteolysis? 

(A question that also remains in my head as well is, the second clear as daylight band up above, could it be the enterokinase that I used to cleave the tags with? Enterokinase has a weight of around 26 kDa that, according to the datasheet, appears to be 31 kDa on SDS, which would pretty much fit). But i digress

Oh, you got me wrong... What I meant was about the two very close bands near your protein. I believe near the protein you have two bands, one with aproximately 76 kDa and other with aproximately 95kDa... Maybe (and it's just a guess), they correspond to your protein, with and without tag (enterokinase activity might have been incomplete). 

The band that is downstream, isolated and well defined... I would say is the enterokinase...

No worries Rafael, I got it no problem, and honestly thanks so much for your input. I just don't think it was the tags in that 'double band' because these were very light (as i said around 17kd).

Possible indeed that some other protein was in that smudged 'band'. The idea has been growing in my head. The MWCO I used (nothing else available on the lab that day) was 50,000Da. That is not 50% smaller than a possible 95kDa contaminant.

Thanks again

This is an interesting story but somewhat unclear:  

Is the photograph a Western blot or a  stained gel?  If a Western blot then why is the putative enterokinase being detected or is there a lane with enterokinase alone?  Was this a NuPage gel with reducing agent in the cathode chamber?  Has a gel been run reduced versus non-reduced done in side-by-side lanes?   Is the expression system eukaryotic or prokaryotic?  Is the amino acid sequence known providing number of cysteines and potential N or O glycosylation sites?  Regarding clipping of tags, are the three tags constructed tandomly  in a train or one or more is at the N-terminus while the other(s) is at the C-terminus?  Has a Western blot been done for any of the tags?  The molecular weight standards seem to be running normally, your positive control for any gel issues, but were the standards prepared in the exact same way as the sample even with TCEP?  Indeed, I've seen smears like this before and the answers to the questions will help to explain the gel pattern in the picture you sent.