I have used Antibody sc-133607 as an example here . According to the company sample of WB in cell line it gives two bands bigger than 90kD and 144kD the latter I suppose is the predicted size of protein according to the protein database ( I don't have any idea why a single protein should appear on blot with two bands? Could this be isoforms, modification..? ).
Literature review is of least help because people often have developed homemade Ab in their labs. The challenge is that I hardly can see that band near to predicted size on my mouse samples that seems strange to me (picture is awful after long exposure) and there are also multiple sharp bands between 90 to 130kd. I was curious first if anybody has used this Ab before or generally how we should explain our results while a polyclonal gives us such complicated patterns? Or should I simply try various Ab from different companies? (not budget friendly anyway!) Any ideas?
Before trusting any western blot signal you should confirm the identity of the band by knockdown or knockout. For a protein like this that is not studied extensively, you might want to order a few different antibodies from different companies and then send back the ones that don't work. If siRNA is difficult to transfect in your cell line, you will probably want to use shRNA lentiviruses like those sold here: http://www.genecopoeia.com/product/search/detail.php?prt=11&cid=&key=MSH033757
Are there different splicing variants of your protein or post-translational modifications such as glycosylation? This could explain the different sizes. Are you sure your cells do express the protein you are looking for? In addition to a knockdown/knockout approach I would definitively include a positive control (cells you are sure they express high amonts or recombinant protein).
monocloanls recognized only a single epitope in an antigen (but: have a look for repetitive sequences, polymers, ... ),
polyclonals recognize several epitopes so that you can find your antigen also at different positions (MW, IP, ...) due to different splicings, posttranslational modifications, precursors, digested fragments, ...
YOu should check all this opportunities. The best way to avoid a proteolytic digestion (which is often present) is to take in your sample a protease inhibitor (e.i. PMSF) or put your sample directely into the sample buffer. The SDS will inactivate also all proteases by denaturations. To inhibit the proteolytic digestion already in the cell culture is much more difficult. Compare supernatants vs. washed and lysed cells. Here you can find also precursoers, different splicing products, ...
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