Thanks for the replies. With regards to protein folding, we run SDS-PAGE under denaturing conditions, and boil the samples prior to loading. Apart from the gel, the plant protein extraction buffer itself comprises many of the same components as standard gel loading buffers (4% SDS, 8 M urea, 50 mM Tris-HCl, 5% BME, 20% glycerol). Recently we also tried out an unmasking buffer (2% SDS, 62.5 mM Tris-HCl, 100 mM BME) directly on the blot, but we didn't observe a band even for the bacterial pepB antigen that we use as a positive control. So, that protocol will require a fair bit of optimization.
On that note: we always load the bacterial antigen alongside our transgenic samples, and yes, anti-pepB detects bacterial-produced pepB just fine.
Sorry I wasn't clear about the animal immunizations. Turns out I was a little confused myself, since the ELISAs were conducted by a former colleague almost a year ago. It turns out that the plates were coated with only a linear epitope of pepB, not the whole pepB antigen itself, and is therefore inconclusive. (That said, ELISA plates coated with bacterial pepA antigen react very nicely to the sera from immunized mice -- so that part of the fusion protein seems to be working alright.)
We're now thinking of cloning the exact same plant codon-optimized sequence into a bacterial expression vector; hopefully codon preference won't be a deterrent. If the bacterial pepA-pepB fusion protein is recognized by anti-pepB Ab, we'll know that the issue is related to possible PTM mechanisms in the plant system. Maybe then we can use the bacterial fusion protein as an immunogen to raise our own antibodies in rabbits. If the bacterial version isn't recognized either, however... we're back to square one.
As far as antibodies are concerned, we've only tried a polyclonal from Abcam thus far. Other commercial vendors do supply it, but in general the prices are a bit too steep for us to play trial and error. The full-length pepB protein has never been expressed in plants before, so trying to figure out what works and what doesn't is proving to be a challenge.
At this stage it no longer matters whether plant pepB is immunogenic or not -- I'll just be happy with a positive Western Blot to corroborate our mass spec findings and prove that pepB is, in fact, being expressed.